ACTA s AGRiCULTURAE SLOVENICA Biotehniška fakulteta Univerze v Ljubljani Biotechnical Faculty University of Ljubljana Acta agriculturae Slovenica • ISSN 1581-9175 • 115 - 2 • Ljubljana, junij 2020 115-2 vsebina.indd 241 23. 06. 2020 07:25:15 115-2 vsebina.indd 242 23. 06. 2020 07:25:15 Acta agriculturae Slovenica Volume / Letnik 115 • Number / Številka 2 • 2020 VSEBINA / CONTENTS Antonio AGUILAR-LOPEZ, Salvador GONZÁLEZ-ANDRADE, Aleš KUHAR 215 Estimation of Engel curves for household expenditure on dry bean and processed bean in Mexico Ocena Engelovih krivulj za izdatke gospodinjstev za suhi in predelani fižol v Mehiki Mojca KOROŠEC, Jasna BERTONCELJ 223 Pomen čebeljih pridelkov v humani prehrani The importance of bee products in human nutrition Vaibhav Bhagwan AWACHAT, Arumbackam Vijayarangam ELANGOVAN, Olajide Mark SOGUNLE, Corbon Godfrey DAVID, Jyotirmoy GHOSH, Shivakumar Nisarani Kollurappa GOWDA, Subrat Kumar BHANJA, Samir MAJUMDAR 237 Influence of in ovo and pre-starter zinc and copper supplementation on growth performance and gastrointestinal tract development of broiler chickens Vpliv dodatka cinka in bakra v jajce in v krmo po izvalitvi na rast in razvoj prebavil pri brojlerskih piščancih Babasola Daniel ADEWALE and Beatrice Abanum NDUKA 247 Genotype and within-pod bean position microenvironment effect on seed choice for raising cocoa (Theobroma cacao L.) seedlings Genotip in mikrookolje glede na položaj semena znotraj ploda vplivata na izbor semen kakavovca (Theobroma cacao L.) za vzgojo sadik Ildar GANEEV, Khasan KARIMOV, Shamil FAYZRAKHMANOV, Ilgam MASALIMOV, Valeri PERMYAKOV 261 Intensification of the drying process of small seed oilseeds using microwave electromagnetic radiation Pospeševanje sušenja majhnih semen oljnih poljščin z mikrovalovnim elektromagnetnim sevanjem Khaled A.A. ABDELAAL, Sahar H. RASHED, Adel RAGAB, Akbar HOSSAIN, Ayman EL SABAGH 273 Yield and quality of two sugar beet (Beta vulgaris L. ssp. vulgaris var. altissima Doll) cultivars are influenced by foliar application of salicylic acid, irrigation timing, and planting density Vpliv foliarnega dodajanja salicilne kisline, časa namakanja in gostote setve na pridelek in kakovost dveh sort sladkorne pese (Beta vulgaris L. ssp. vulgaris var. altissima Doll) Insha YOUSUF and Abdul A. BUHROO 283 Seasonal incidence and bionomics of rose aphid, Macrosiphum rosae (Linnaeus, 1758), (Hemiptera: Aphididae) in Kashmir, India Sezonsko pojavljanje in bionomika vrtnične uši (Macrosiphum rosae (Linnaeus, 1758), Hemiptera: Aphididae) v Kašmirju, Indija Seda TUNÇAY ÇAGATAY, Gûlçah ÇALIK KOÇ, Fereshteh REZAEl, Ozlem DARCANSOY ΧERI, Feride Iffet §AHIN, Mehmet HABERAL 297 Evaluation of production conditions of tomato grafted with different tobacco rootstocks and determining nicotine content and quality of fruit Ovrednotenje pridelovalnih razmer paradižnika cepljenega na različne podlage tobaka in določitev vsebnosti nikotina in kakovosti plodov Asgar EBADOLLAHI 307 Estragole-rich essential oil of summer savory (Satureja hortensis L.) as an eco-friendly alternative to the synthetic insecticides in management of two stored-products insect pests Na estragolu bogato eterično olje vrtnega šetraja (Satureja hortensis L.) kot okolju prijazna alternativa sintetičnim insekticidom pri zatiranju dveh vrst skladiščnih škodljivih žuželk 115-2 vsebina.indd 243 23. 06. 2020 07:25:16 315 329 341 349 357 369 377 389 399 409 417 429 437 244 Pooran GOLKAR, Nasrin RAHMATABADI, Seyyed Ali Mohammad MIRMOHAMMADY MAIBODY Improvement of yield and yield stability in safflower using multivariate, parametric and non-parametric methods under different irrigation treatments and planting date Izboljšanje pridelka žafranike in njegove stabilnosti z multivariatnimi parametričnimi in neparametričnimi metodami pri različnem namakanju in datumih setve Jemal MOHAMMED, Wassu MOHAMMED, Eleni SHIFERAW Correlation and path coefficient analysis among agro-morphological and biochemical traits of okra [Abelmoschus esculentus (L.) 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BUHROO, Abdul Lateef KHANDAY Seasonal incidence of apple leaf miner (Lyonetia clerkella (L., 1758), Lepidoptera, Lyonetiidae) in Kashmir, India Sezonsko pojavljanje sadnega listnega zavrtača (Lyonetia clerkella (L., 1758), Lepidoptera, Lyonetiidae) v Kašmirju, Indija Nwakuche Chinenye ONWUBIKO, Michael Ifeanyi UGURU, Grace Ovute CHIMDI Pattern of variation and grouping of qualitative morphological characters of bambara groundnut (Vigna subterranea (L.) Verdc.) Vzorec spreminjanja in združevanja morfološki znakov bambare (Vigna subterranea (L.) 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Vuill. in eteričnih olj na bombaževo uš (Aphis gossypii Glover, 1877, Hemiptera: Aphididae) Amir ABDoLLAHI, Masoud sHAFAFI ZENooZIAN, Mohammad Reza sAEIDIAsL, Mohammad ARMIN, Adel BEIGBABAEI 473 Modeling the chemical properties of sesame oil under the influence of pulsed electric field using the artificial neural networks Modeliranje kemijskih lastnosti sezamovega olja pod vplivom pulzirajočega električnega polja z uporabo umetnih nevronskih mrež Jože MAČEK 485 Delovanje dunajskega Terezianuma in Theodorja Kravine na področju ekonomije in agronomije Activity of Vienna Terezianum and Theodor Kravina on the field of economics and agronomy Nik sUsiČ, saša ŠIRCA, Gregor UREK, Barbara GERIČ sTARE 495 Senecio vulgaris L. recorded as a new host plant for the root-knot nematode Meloidogyne luci Dokumentiranje nove gostiteljske rastline Senecio vulgaris L. za rastlinsko parazitsko ogorčico Meloidogyne luci Muhammad Aamir IQBAL 499 Ensuring food security amid novel coronavirus (COVID-19) pandemic: Global food supplies and Pakistan's perspectives 503 Navodila avtorjem Author guidelines 115-2 vsebina.indd 245 23. 06. 2020 07:25:16 115-2 vsebina.indd 246 23. 06. 2020 07:25:16 doi:10.14720/aas.2020.115.2.1415 Original research article / izvirni znanstveni članek Estimation of Engel curves for household expenditure on dry bean and processed bean in Mexico Antonio AGUILAR-LOPEZ \ Salvador GONZALEZ-ANDRADE 2, Aleš KUHAR 3 4 Received January 06, 2020; accepted May 04, 2020. Delo je prispelo 06. januarja 2020, sprejeto 04. maja 2020. Estimation of Engel curves for household expenditure on dry bean and processed bean in Mexico Abstract: Dry bean is the leading source of low-cost plant-based proteins in Mexico. However, in the years following the liberalization of the economy, Mexico experienced the erosion of a self-sufficiency index for this commodity. Impending changes in the international markets for proteins compel us to reevaluate the role of dry bean for Mexico's food security. In the present paper we set out to analyze the last link of the marketing chain in Mexico's dry bean market: the consumer. Using data on household expenditure for 2018, the relationship between income and expenditure on dry bean as well as on processed bean is ascertained by means of the Working-Leser Engel Curve equations system. Due to the presence of zero-expenditure households in the sample, we followed the two-step Heckit procedure for the possible selection bias. The results suggest that the budget share for dry bean and for processed bean drops as income increases. The corrected conditional elasticity for dry bean is -0.1056. For processed bean, the elasticity is -0.2286. The negative sign indicates that both commodities are inferior goods. Key words: plant production; plant based proteins; dry bean; economics; Engel curves; household income; food self-sufficiency; Mexico Ocena Engelovih krivulj za izdatke gospodinjstev za suhi in predelani fižol v Mehiki Izvleček: Suhi fižol je v Mehiki najpomembnejši cenovno ugoden vir rastlinskih beljakovin, vendar je Mehika v letih po liberalizaciji gospodarstva doživela padec indeksa samooskrbe za to proizvodno skupino. Zaradi bližajočih se sprememb v ureditvi mednarodne trgovine, ki bodo vplivale tudi na trgovanje z beljakovinsko bogatimi kmetijskimi surovinami, smo želeli ponovno oceniti pomen suhega fižola za prehransko varnost v Mehiki. V pričujočem prispevku smo analizirali zadnji člen tržne verige za suhi fižol na mehiškem trgu, potrošnika. Z uporabo podatkov o izdatkih gospodinjstev za leto 2018 smo razmerje med dohodkom in odhodki za suh in predelan fižol ugotavljali z uporabo Working-Leser-jevega sistema Engelovih krivulj. Zaradi prisotnosti gospodinjstev, ki niso imela tovrstnih stroškov, smo upoštevali dvostopenjski Heckit-ov postopek za korekcijo morebitne napake pri vzorčenju. Rezultati kažejo, da se delež proračuna za suh in predelan fižol zmanjšuje, ko se dohodek gospodinjstev povečuje. Korigirana pogojna elastičnost za suhi fižol je -0.1056. Za predelan fižol je elastičnost -0,2286. Negativni predznak potrjuje status inferiornih dobrin za obe proučevani kategoriji. Ključne besede: poljščine; rastlinske beljakovine; suhi fižol; ekonomika; Engelove krivulje; dohodki gospodinjstev; pre-hranska varnost; Mehika 1 Instituto Tecnológico Superior de Huichapan, División de Ingeniería en Gestión Empresarial, Huichapan, México 2 El Colegio de la Frontera Norte, Departamento de Estudios Económicos, Tijuana, México 3 University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Ljubljana, Slovenia 4 Corresponding author, e-mail: ales.kuhar@bf.uni-lj.si Acta agriculturae Slovenica, 115/2, 215-222, Ljubljana 2020 AAS_vsebina_202Q_115_2_ZOOT_110520.indd 215 16. 06. 2020 09:47:57 A. AGUILAR-LOPEZ et al. 1 introduction A notion echoed in several academic and institutional settings around the world suggests that plant-based proteins should account for a larger share of the human intake of these nutrients, replacing animal-based sources to some extent. This partial replacement is seen as an economic and environmental necessity, feasible in nutritional terms, and strategically unavoidable for the achievement of food security goals at the national and international levels. The strategic dimension is especially significant in the Developing Countries, which rely more on plant-based sources of protein (Grigg, 1995). Although there is still uncertainty about the degree whereby climate change and the depletion of natural resources could threaten the capacity to sustain the rates of growth in agricultural output observed during the previous decades (Valin et al., 2014), the expectation is that the prices of grains and meat will increase in the long-run even if global food supply increases (Aiking, 2011). Although average meat consumption stagnates and even declines as income increases (Vranken et al., 2014), different strategies have been suggested to adapt human diets to meet sustainability challenges (De Boer et al., 2014), one among them being the substitution of animal-based proteins for plant-based proteins (Westhoek et al., 2014). The instrumental use of pulses in a range of areas, namely: food security, nutrition, health, sustainable agriculture and climate mitigation, is such that 2016 was declared the International Year of Pulses by the United Nations General Assembly (Calles et al., 2019). However, to fully harness these potentials, actions need to be taken in order to reverse the decline in consumption of pulses witnessed worldwide (implying a change, yet again, in consumer preferences), to encourage production (which currently takes place in marginal areas) and to further the development of their marketing chains. The present paper is an attempt at characterizing the last link of Mexico's dry bean marketing chain, i.e., consumption, as well as other factors that ought to be considered when designing policies aimed at tackling challenges in the areas mentioned before. Dry bean is an important source of protein, among other nutrients, in the human food supply; in fact, dry bean contains 15-25 % of protein on a dry weight basis, depending on the variety (Sathe, 2002). In Mexico, this pulse is one of the leading sources of plant-based proteins, it is the second annual crop by planted area, and it has a historical relationship with the inhabitants of the country, which is deemed as the center of origin of a number of varieties. Four factors that could affect food security regarding the availability and prices of proteins at the national level are: a) changes in agricultural productivity levels due to climate change (Parry et al., 2004), b) the conflict between animal-based sources and plant-based sources for the natural resources required for their production, as well as the interaction between their production processes and the so-called planetary boundaries (Stehfest et al., 2009), c) changes in the consumption pattern of proteins in countries such as China and India, due to income and population growth (Gandhi & Zhou, 2014), and d) the trans- Figure 1: Components and tendency of Mexico's self-sufficiency index for dry bean (1986-2017); quantities in thousand metric tons. Source: own elaboration with data from FAO, 2019) 216 Acta agriculturae Slovenica, 115/2 - 2020 AAS_vsebina_2020_115_2_ZOOT_110520.indd 216 16. 06. 2020 09:47:57 Estimation of Engel curves for household expenditure on dry bean and processed bean in Mexico Table 1: Average budget share (%) for dry bean and processed bean by income decile (quarterly data), 2008-2018 Income decile 2 ENIGH 1 I 3 II III IV V VI VII VIII IX X Code 2008 3.590 1.507 1.023 0.854 0.616 0.517 0.376 0.277 0.174 0.084 A137 2010 3.920 1.586 1.066 0.774 0.527 0.516 0.425 0.262 0.161 0.067 Dry bean 2012 3.198 1.519 1.384 0.900 0.711 0.532 0.421 0.286 0.176 0.087 2014 2.389 1.220 0.831 0.708 0.519 0.420 0.305 0.238 0.152 0.070 2016 2.216 1.148 0.792 0.624 0.490 0.387 0.306 0.230 0.150 0.072 2018 2.245 1.026 0.715 0.540 0.452 0.340 0.284 0.210 0.136 0.065 2008 0.155 0.120 0.063 0.082 0.065 0.051 0.046 0.029 0.027 0.020 A142 2010 0.099 0.106 0.077 0.075 0.049 0.056 0.049 0.036 0.024 0.016 Proc. bean 2012 0.187 0.127 0.096 0.105 0.086 0.076 0.057 0.042 0.046 0.011 2014 0.205 0.108 0.066 0.068 0.060 0.057 0.053 0.050 0.032 0.024 2016 0.183 0.138 0.117 0.100 0.093 0.088 0.068 0.057 0.046 0.022 2018 0.210 0.152 0.127 0.110 0.100 0.086 0.079 0.060 0.048 0.024 1 Following the procedure whereby the expenditure on other foodstuffs reported in the 'concentrado' tables were obtained. 2 The expansion factor was used when determining the income deciles. 3 Budget share for households with no income was set to 0 for decile I. Source: own elaboration with data from INEGI, 2019a. mission of price spikes from international to domestic markets (Bekkers et al., 2017). World production of dry beans grew steadily between 1986 and 2017, period in which it went from 17,1 to 31,4 million tons. Mexico ranked among the top ten world producers during this interval; however, its production level didn't follow this upward trend. In fact, Mexico's domestic production averaged 1,11 million tons during the same period, with significant year-over-year variations. At the same time, dry bean imports (mainly from the U.S.) showed a positive trend, but so did the relatively less significant exports (FAO, 2019). On the other hand, Mexico's population went from 81.2 million in 1990 to 119.9 million in 2015 (INEGI, 2019b). When analyzing these trends, it can be inferred that per capita consumption of dry bean among Mexicans dropped in the years after the liberalization of the economy started; yet, Mexico witnessed the slightly eroding tendency of a self-sufficiency index for Figure 2: Average budget share for dry bean and processed bean (%) for the lower income deciles (quarterly data), 2008-2018. Source: own elaboration with data from INEGI 2019a Acta agriculturae Slovenica, 115/2 - 2020 217 AAS_vsebina_2020_115_2_ZOOT_110520.indd 217 16. 06. 2020 09:47:57 A. AGUILAR-LOPEZ et al. Table 2: Average budget share (%) for animal-based foodstuffs and non-processed pulses by income decile (quarterly data), 2008-2018 Income decile 2 ENIGH 1 I 3 II III IV V VI VII VIII IX X Code 2008 14.440 10.333 8.669 7.903 7.436 6.784 5.485 4.624 3.811 2.378 Animal 4 2010 16.202 10.856 9.683 8.284 7.525 6.931 6.037 5.131 4.173 2.575 Protein 2012 15.994 10.556 10.084 8.324 8.137 7.020 6.597 5.265 4.314 2.700 2014 14.661 10.650 10.000 9.204 8.412 7.360 6.716 6.060 4.776 3.026 2016 13.797 10.155 8.960 8.269 7.450 6.444 6.140 5.154 4.229 2.754 2018 13.496 9.902 8.969 8.052 7.481 6.653 6.043 5.200 4.380 2.804 2008 0.188 0.108 0.100 0.065 0.072 0.053 0.040 0.031 0.017 0.012 Pulses 2010 0.219 0.125 0.108 0.073 0.074 0.056 0.040 0.036 0.025 0.012 Non-proc. 2012 0.160 0.113 0.106 0.078 0.048 0.075 0.040 0.034 0.020 0.012 2014 0.121 0.147 0.109 0.079 0.088 0.074 0.040 0.034 0.030 0.012 2016 0.198 0.120 0.102 0.081 0.061 0.055 0.041 0.031 0.022 0.012 2018 0.172 0.116 0.098 0.072 0.068 0.063 0.048 0.038 0.027 0.015 1 Following the procedure whereby the expenditure on other foodstuffs reported in the 'concentrado' tables were obtained. 2 The expansion factor was used when determining the income deciles. 3 Budget share for households with no income was set to 0 for decile I. 4 Animal protein = carnes + huevo + pescado, from the 'concentrado' tables. Source: own elaboration with data from INEGI, 2019a. this commodity (measured as the domestic production to apparent national consumption ratio) (Figure 1), since the increase in the domestic demand (driven by population growth) was met by increasing imports. Among the factors that influence the spatial variation in the quantity and sources of protein consumed, income and cost stand out, as well as the effect of the environmental conditions on the selection of staple crops at the local level (Grigg, 1995). In the paper at hand, we set out to examine Engel's law (i.e. the principle stating that poorer households dedicate a higher share of their income to food than richer households), applied to both dry bean and processed bean. In economics, the so-called Engel curves relate expenditure on different commodities and income at the household level. The latest data available on household expenditure and income in Mexico, is provided by the 2018 national survey 'Encuesta Nacional de Ingresos y Gastos de los Hogares' (ENIGH). Figure 3: Average budget share for animal-based foodstuffs and non-processed pulses (%) for the lower income deciles (quarterly data), 2008-2018. Source: own elaboration with data from INEGI 2019a 218 Acta agriculturae Slovenica, 115/2 - 2020 AAS_vsebina_2020_115_2_ZOOT_110520.indd 218 16. 06. 2020 09:47:58 Estimation of Engel curves for household expenditure on dry bean and processed bean in Mexico Table 1 displays the budget share (i.e. the expenditure to income ratio) for both dry bean and industrialized bean across income deciles (quarterly current income). Engel's law seems to hold in the two cases. However, a closer look at the evolution of household expenditure reveals that households in the lower income deciles tend to spend far more on dry bean than on processed bean (Figure 2). The analysis of household expenditure on dry bean is supplemented with an analysis of the expenditure on other sources of proteins. Table 2 displays the budget share for both animal-based sources of proteins (eggs, fish, and meat) and non-processed pulses (chickpeas, lentils, lima beans, and peas), during the period 2008-2018. Furthermore, the Figure 3 shows the average budget shares for animal-based foods and non-processed pulses for the lower four income deciles in the ten years' period. 2 materials and methods The 2018 ENIGH contains a set of zero-expenditure households for both dry bean and processed bean (Table 3). In single-equation representations of Engel curves, the Heckman (or Heckit) two-step procedure has been used to circumvent this censored-response problem (Saha et al., 1997). The Heckman estimation method of Engel curves is based on the idea that censored data on household expenditure can be seen as a combination of a selection Table 3: Descriptive statistics of the expenditure variables used in the analysis Pr[Zi = l|Wj] = 0 or equivalently where z. = 1. In the paper at hand, the equation of the selection mechanism is given by: zi=y1+ y2ln(income) + ^ YkWk + (5) k=3 Code Commodity % of non-zeros Mean 1 Std. Dev. Min Max A137 Dry bean 33.24 138.80 256.87 0.00 6,428.57 A142 Processed bean 11.81 37.28 129.47 0.00 4,049.96 1 Mexican Pesos. Source: own elaboration with data from INEGI, 2019a. Some functional forms used to examine Engel's law include: double logarithmic, quadratic, semi-logarithmic and Working-Lesser. The latter approach relates budget share y. and the logarithm of income; it also allows a direct test of En-gel's law (Holcomb et al., 1995). The Engel curve used in the second step of the Heckit follows the Working-Lesser form defined by: mechanism for the decision to purchase and a model for the level of the expenditure, which applies only to Ji the sample of households with actual expenditure. The procedure starts off with the equation that determines the sample selection: Zi = w'iY + Ui (1) Where zi is an indicator variable equal to 1 if expenditure occurs in household i and 0 otherwise, w. is a vector of observed socio-demographic characteristics of the household that affect the purchase decision, and y is a vector of coefficients which is determined by the Maximum Likelihood (ML) estimation of a probit e, = 1 + -model where: = Pi+ P2ln{income{) + ^ pkxKi + GXi + Et (6) k=3 This analysis is supplemented with a corrected estimate of the income elasticity of the budget share given by (at the mean of the data): P2 + dE (7) Where w2 = ln(income). Which is equivalent to (Saha et al., 1997): ^ [02 - Qy2 {£«y)£(10 + E^f}] (8) Acta agriculturae Slovenica, 115/2 - 2020 219 AAS_vsebina_2020_115_2_ZOOT_110520.indd 219 16. 06. 2020 09:47:58 A. AGUILAR-LOPEZ et al. The data used in this analysis is provided by Mexico's Instituto Nacional de Estadística y Geografía and are representative at the national level. The 2018 ENIGH provides with a sample of 74,647 households in a table labeled as 'concentradohogar', with records on their expenditure on selected food groups, as well as their socio-demographic features, including size and income. In this analysis, the income variable corresponds to quarterly current income. The expenditure on dry bean was obtained following the same procedure used to get the expenditures on the food groups recorded in the 'concentradohogar' table, i.e. as the summation of the quarterly expenditure (gasto trimestral) on the code A137 from the 'gastoshog-ar' table plus the summation of the quarterly expenditure on the same code from the 'gastospersona' table. For the expenditure on processed bean, we replicated the procedure using the code A142. In order for the budget shares to be confined between zero and one, households that reported having either no-income or expenditure on dry bean greater than income were removed from the sample. This rendered a subset of 74,637 households. The sociodemographic characteristics considered in this paper are: 1) income (IngCor); 2) size of locality (TamLoc); 3) region (Region); 4) household class (Clase-Hog); 5) education of the head of the household (Educa-Jefe); 6) sex of the head of the household (SexoJefe); 7) age of the head of the household (EdadJefe); 8) number of grown-ups (Mayores); 9) size of the household (TotIn-teg); and 10) socio-economic strata (EstSocio). We added dummy variables to indicate expenditure on: 11) dry bean (A137_dum); 12) processed bean (A142_dum); 13) meat products (Carnes_dum); 14) eggs (Huevo_dum); and 15) fish (Pescado_dum). The levels for size of locality (number of inhabitants) are: 100,000 and more = 1; 15,000-99,999 = 2; 2,500-14,999 = 3; 2,500 and less = 4. The region variable is a categorical one with the following levels: NW (Baja California, Baja California Sur, Chihuahua, Durango, Sinaloa, and Sonora); NE (Coa-huila, Nuevo León, and Tamaulipas); W (Colima, Jalisco, Michoacán, and Nayarit); E (Hidalgo, Puebla, Tlaxcala, and Veracruz); CN (Aguascalientes, Guanajuato, Queré-taro, San Luis Potosí, and Zacatecas); CS (Ciudad de México, Estado de México, and Morelos); SW (Chiapas, Guerrero, and Oaxaca); and SE (Campeche, Quintana Roo, Tabasco, and Yucatán). The education of the head of the household ranges from: Without instruction = 1, to Graduate = 11. The levels for sex of the head of the household are: Male = 1; and Female = 2. The levels for socioeconomic strata and household class are inherited from the ENIGH terminology. The socioeconomic strata are: Lower = 1; Lower middle = 2; Upper middle = 3; and Upper = 4. Finally, the household classes are: One-person = 1; Nuclear = 2; Extended = 3; Composite = 4; and Coresident = 5. 3 results Table 4 shows the OLS Engel curve estimates for dry bean and processed bean for Mexico, using the 2018 data. It also shows the ML for the probit model used in the first stage of the Heckit procedure for dry bean. The results obtained show that the coefficient associated with the IMR is significant only for dry bean, which indicates that the correlation between the error term of the decision to purchase this commodity and the budget share of the same is different than zero. The use of the Heckit procedure is appropriate in the case of dry bean; therefore, household expenditure on this commodity can be represented as a two-stage process. The variables included in the probit model were determined on the basis of the Akaike Information Criterion (AIC), by step-wise regression. Regarding the decision to purchase dry bean, the income variable is statistically significant. This result indicates that income affects negatively the probability of purchasing this commodity. The effect of household size is positive and statistically significant, which indicates that having a larger household increases the propensity to spend on dry bean. In order to determine the final form of the Working-Lesser structure in the second step of the Heckit procedure, we tested for collinearity in a model with the same regres-sors used for the probit in the first stage, plus the IMR. Then, we removed the variable with the highest variance inflation factor (VIF) keeping the IMR, so that in the final model all VIF's fell below the cut-off value of 10. Thus, the variables in the OLS model for dry bean are a subset of the variables included in the probit model. Both OLS models from Table 4 exhibit heteroske-dasticity. It has been pointed out that the standard errors and the heteroskedasticity-robust standard errors of the OLS estimates provided by the second stage of the Heckit models, are incorrect. Although formulas to overcome this problem are available, their implementation is not always easy; however, one alternative is to use bootstrapped standard errors (Cameron & Trivedi, 2005, p. 550). We followed this approach for dry bean, whereas in the case of processed bean, we present heteroskedasticity-robust standard errors. The Working-Lesser structure reported negative and statistically significant parameter estimates for the logarithm of current income for dry bean and processed bean. The corrected conditional elasticity estimated at the mean of the data for dry bean is -0.1056. Whereas, the 220 Acta agriculturae Slovenica, 115/2 - 2020 AAS_vsebina_2020_115_2_ZOOT_110520.indd 220 16. 06. 2020 09:47:58 Estimation of Engel curves for household expenditure on dry bean and processed bean in Mexico Table 4: Heckit and OLS estimates of the Engel curve for dry bean and for processed bean Dependent variable: 1St SteP: ZiA137 2nd ^p: yiA„7 y i,A142 Regressor Probit-ML OLS OLS Log(IngCor) *** _ V -0.1847 (0.0088) *** , V -0.0212 (0.0005) -0.0135*** (0.0012) TamLoc = 2 0.0912*** (0.0176) -0.0002 (0.0003) TamLoc = 3 0.1401*** (0.0181) -0.0004 (0.0003) TamLoc = 4 0.1322*** (0.0164) 0.0013*** (0.0003) Region = NW *** , -0.0957 (0.0169) 0.00004 (0.0004) 0.0014*** (0.0004) Region = SE *** , 0.1243 (0.0199) *** , -0.0034*** (0.0004) -0.0030*** (0.0004) Region = SW -0.0152 (0.0203) *** , 0.0022*** (0.0006) 0.0002 (0.0005) Region = CS -0.0145 (0.0207) -0.0004 (0.0005) -0.0027*** (0.0005) Region = NE *** , -0.1725 (0.0200) 0.0010* (0.0006) 0.0002 (0.0007) Region = W 0.0271 (0.0199) -0.0006 (0.0004) -0.0002 (0.0005) Region = E 0.0791*** (0.0196) -0.0013*** (0.0005) -0.0032*** (0.0005) ClaseHog = 2 0.0973*** (0.0253) -0.0039*** (0.0009) ClaseHog = 3 0.0338 (0.0306) -0.0040*** (0.0010) ClaseHog = 4 0.0240 (0.0687) -0.0024 (0.0016) ClaseHog = 5 -0.3212*** (0.1169) -0.0002 (0.0023) EducaJefe = 2 -0.0630 (0.1505) -0.0028 (0.0032) -0.0006 (0.0040) EducaJefe = 3 -0.0249 (0.0216) *** , -0.0025*** (0.0007) -0.0027** (0.0012) EducaJefe = 4 *** , -0.0663*** (0.0223) *** y -0.0034*** (0.0007) -0.0026** (0.0013) EducaJefe = 5 *** , -0.1081 (0.0336) *** , -0.0035*** (0.0009) -0.0028** (0.0014) EducaJefe = 6 *** , -0.1385 (0.0226) *** , -0.0040*** (0.0007) -0.0025** (0.0012) EducaJefe = 7 *** , -0.2427*** (0.0359) *** , -0.0040*** (0.0008) -0.0033*** (0.0012) EducaJefe = 8 -0.2225*** (0.0265) -0.0030*** (0.0007) -0.0012 (0.0016) EducaJefe = 9 -0.3497*** (0.0414) -0.0005 (0.0009) -0.0017 (0.0013) EducaJefe = 10 -0.2934*** (0.0296) 0.0023*** (0.0008) -0.0004 (0.0014) EducaJefe = 11 -0.3185*** (0.0524) 0.0073*** (0.0012) 0.0038** (0.0018) SexoJefe = 2 -0.0013*** (0.0004) Log(EdadJefe) 0.1132*** (0.0205) 0.0028*** (0.0005) 0.0021*** (0.0004) Log(Mayores) *** , 0.2122 (0.0230) *** , 0.0033*** (0.0006) Log(Totlnteg) *** , 0.2248*** (0.0224) 0.0041 (0.0006) 0.0031*** (0.0006) EstSocio = 2 *** , -0.2147 (0.0147) *** , -0.0044*** (0.0003) 0.00002 (0.0007) EstSocio = 3 -0.3129*** (0.0230) -0.0028*** (0.0005) 0.0002 (0.0009) EstSocio = 4 -0.3531*** (0.0311) 0.0007 (0.0006) 0.0022** (0.0010) A142_dum = 1 -0.5798*** (0.0176) -0.0025*** (0.0005) A137_dum = 1 -0.0030*** (0.0003) Carnes_dum = 1 0.4133*** (0.0143) -0.0037*** (0.0006) -0.0019*** (0.0007) Huevo_dum = 1 0.4305*** (0.0112) 0.0005 (0.0005) Pescado_dum = 1 0.1379*** (0.0130) 0.0011*** (0.0003) 0.0006 (0.0005) A137_mr 0.0019** (0.0010) Constant 0.1617 (0.1199) 0.2270*** (0.0047) 0.1449*** (0.0115) Observations 74,637 24,808 8,818 R2 0.4041 0.2674 Log Likelihood -41,773.2200 Akaike Inf. Crit. 83,618.4300 Residual Std. Error 0.0192 (df = 24772) 0.0148 (df = 8789) F Statistic 479.9184*** (df = 35; 24772) 114.5816*** (df = 28; 8789) Notes: Standard errors in parentheses; in the case of dry bean, bootstrapped estimates after 3,000 samples; in the case of processed bean, heteroske-dasticity-robust. Significance levels: *p < 0.1; **p < 0.05; ***p < 0.01. Own elaboration with data from INEGI, 2019a. Acta agriculturae Slovenica, 115/2 - 2020 221 AAS_vsebina_2020_115_2_ZOOT_110520.indd 221 16. 06. 2020 09:47:58 A. AGUILAR-LOPEZ et al. elasticity for processed bean is -0.2286. The negative sign indicates that both commodities are inferior goods. 4 conclusions The use of pulses for the attainment of policy objectives in several areas demands the reversing of the downward trend in consumption that these commodities have displayed worldwide. In the present paper, the strategic importance of dry bean for Mexico is pointed out; this pulse remains as the leading source of plant-based proteins among Mexicans, since it exceeds the expenditure that households make on other pulses. However, a closer look on household expenditure revealed that the above-mentioned trend manifested itself across income deciles during the last decade. To understand the last link of the marketing chain for dry bean in Mexico, we set out to analyze household expenditure on several protein sources, including both dry bean and processed bean. For this purpose, budget share Engel curves were estimated for the two commodities using the 2018 ENIGH sample of households. Our results are in alignment with the principle stated by Engel's law, and both dry bean and processed bean turned out to be inferior goods. Therefore, policy measures aimed at the attainment of environmental, nutritional, and health goals, based on the use of dry bean and other pulses, ought to set out to change the relationship between expenditure on this commodities and income, i.e. turn them into normal goods. One alternative is the advancement of processed versions of these commodities. Other trends worth mentioning in Mexico' market for dry bean are: the slight erosion of a self-sufficiency index in the post-liberalization era of the economy, the decline in the per-capita consumption of this commodity, and the stagnation of the production level (notwithstanding the considerable year-over-year changes). Finally, some of the expected outcomes from policies aimed at furthering dry bean production are: 1) increases in the production levels as more productive land is allocated to this end, and a decline in prices due to higher productivity. 5 references Aiking, H. (2011). Future protein supply. 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Pomen čebeljih pridelkov v humani prehrani Izvleček: Čebelji pridelki so naraven vir hranil in biološko aktivnih spojin, ki se uvrščajo tudi na sezname funkcionalnih sestavin. V prehrani uporabljamo predvsem med in v manjši meri cvetni prah osmukanec in matični mleček. Propolis in čebelji strup se zaradi terapevtskih lastnosti uporabljata predvsem v apiterapiji. Od osnovnih hranil je med predvsem vir sladkorjev, cvetni prah in matični mleček pa poleg teh vsebujeta še beljakovine in maščobe, cvetni prah pa tudi prehran-sko vlaknino. Čebelji pridelki v manjših količinah vsebujejo še bioaktivne spojine, ki imajo antioksidativno, protimikrobno, protivnetno in protivirusno delovanje. Za med so med drugim značilne fenolne spojine, proteini matičnega mlečka, oli-gosaharidi. Matični mleček vsebuje specifične maščobne kisline, vključno z 10-hidroksi-2-decenojsko kislino, bioaktivne peptide, proteine, v cvetnem prahu pa so različni vitamini, fenolne spojine, nenasičene maščobne kisline in druge spojine. Potrebne pa so nadaljnje raziskave in klinične študije za ovrednotenje učinkovitosti čebeljih pridelkov ter ozaveščanje potrošnikov o pomenu njihovega uživanja. Med, cvetni prah osmukanec in matični mleček so naravna živila, ki zaradi svoje sestave lahko pripomorejo k doseganju priporočenih dnevnih vnosov osnovnih hranljivih snovi, hkrati pa so lahko vir pomembnih bioaktivnih spojin, zato nedvomno sodijo v uravnoteženo prehrano človeka. Ključne besede: živila; čebelji pridelki; med; cvetni prah osmukanec; matični mleček; prehrana ljudi; zdravje The importance of bee products in human nutrition Abstract: Bee products are a natural source of nutrients and biologically active compounds, which may also be found on the lists of functional ingredients. In our diets, mainly honey is used and to a lesser extent bee pollen and royal jelly. Propolis and bee venom are mainly used in apitherapy due to their therapeutic properties. Regarding the basic nutrients, honey is primarily a source of sugars, while protein and fat contents are considerable in royal jelly and pollen, which also contains dietary fiber. Bee products also contain small amounts of bioactive compounds that have antioxidant, antimicrobial, anti-inflammatory and antiviral effects. Honey is characterized by, among others, phenolic compounds, royal jelly proteins, oligosaccharides. Royal jelly contains specific fatty acids, including 10-hydroxy-2-decenoic acid, bioactive peptides, major royal jelly proteins, and pollen contains various vitamins, phenolic compounds, amino acids, unsaturated fatty acids. However, further research and clinical studies are needed to evaluate the effectiveness of bee products and to raise consumer awareness of the importance of their consumption. Honey, bee pollen and royal jelly are natural foods, which due to their composition may help to achieve the recommended daily intake of basic nutrients, and may also serve as a source of important bioactive compounds, and therefore undoubtedly belong to a balanced diet. Key words: food; bee products; honey; bee pollen; royal jelly; human nutrition; health 1 Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za živilstvo, Ljubljana, Slovenija 2 Korespondenčna avtorica, e-naslov: mojca.korosec@bf.uni-lj.si Acta agriculturae Slovenica, 115/2, 223-235, Ljubljana 2020 AAS_vsebina_2020_115_2_ZOOT_110520.indd 223 16. 06. 2020 09:47:59 M. KOROŠEC in J. BERTONCELJ 1 uvod Način prehranjevanja lahko pomembno vpliva na zdravje. Naravna živila in naravna prehranska dopolnila imajo v današnjem času pomembno mesto v prehrani ljudi. Del naravnih živil oz. dopolnil predstavljajo tudi čebelji pridelki, med, cvetni prah osmukanec, matični mleček in propolis. Ti pridelki imajo različno vlogo v čebelji družini, zaradi hranilne vrednosti in ugodnih funkcionalnih lastnosti pa se pogosto uporabljajo tudi v prehrani ljudi. Poleg medu, ki je v prehrani poznan že iz pradavnine, in je najbolj uporabljen čebelji pridelek, se v zadnjem času povečuje tudi uporaba cvetnega prahu in matičnega mlečka, slednjega zlasti v obliki prehranskih dopolnil. Propolis in čebelji strup se zaradi terapevtskih lastnosti uporabljata predvsem v apiterapiji (Bogdanov, 2011; Yucel in sod., 2017). Čebelji pridelki imajo visoko biološko vrednost zaradi vsebnosti hranilnih snovi in bioaktivnih spojin. Njihova zastopanost je odvisna od botaničnega in geografskega porekla, podnebnih razmer, postopkov čebelarjenja in skladiščenja čebeljih pridelkov. V vsakdanji prehrani ljudje najpogosteje posegajo po medu, ki ga uživajo samega ali kot sladilo za slajenje pijač in nekaterih drugih živil. Cvetni prah in matični mleček pa se uporabljata predvsem kot dodatek prehrani. Uporabnost čebeljih pridelkov se kaže tudi v možnosti njihovega dodajanja drugim živilom za povečanje vsebnosti bioaktivnih spojin v teh živilih in s tem večje protimikrobne in antioksidativne učinkovitosti (Viuda--Martos, 2008; Cornara in sod., 2017; Pasupuleti in sod., 2017). Prispevek povzema pridobivanje in sestavo čebeljih pridelkov ter možnost uporabe čebeljih pridelkov v vsakdanji prehrani, njihovo aplikacijo v živila in nekatere biološke lastnosti, ki lahko pozitivno delujejo na zdravje človeka. 2 pridobivanje čebeljih pridelkov 2.1 PRIDOBIVANJE MEDU Med je eno najbolj kompleksnih naravnih živil, je naravna sladka snov, ki jo izdelajo čebele Apis mellifera iz nektarja cvetov ali izločkov iz živih delov rastlin ali izločkov žuželk (uši, kaparjev), ki sesajo rastlinski sok na živih delih rastlin, ki jih čebele zberejo, predelajo z določenimi lastnimi snovmi, shranijo, posušijo in pustijo dozoreti v satju. Med pridobivamo iz satovja s centrifugiranjem, brez kakršnekoli obdelave, razen grobega filtriranja. Pravilnik o medu (2011) deli med glede na izvor na i) »med iz nektarja«, ki je pridobljen iz nektarja cvetov različnih rastlin, ter ii) »manin med«, ki je pridobljen predvsem iz izločkov insektov na živih delih rastlin ali izločkov živih 224 Acta agriculturae Slovenica, 115/2 - 2020 delov rastlin. Glede na vrsto paše ločimo različne vrste medu, najpogostejše vrste slovenskega medu so podane v preglednici 1. Če so čebele nabrale nektar ali mano pretežno na eni rastlinski vrsti in med izhaja v celoti ali pretežno iz navedenega izvora, in ima njegove senzorične, fizikalno-kemijske in mikroskopske lastnosti, se ime med lahko dopolni z navedbo oznake, ki se nanaša na izvor iz cvetov ali rastlin (Pravilnik o medu, 2011). Preglednica 1: Vrste slovenskega medu glede na pašo Table 1: Types of Slovenian honey regarding the pasture source Vrsta paše Vrsta medu Nektar akacijev med (Robinia pseudoacacia) med oljne ogrščice (Brassica napus) ajdov med (Fagopyrum esculentum) rešeljikov med (Prunus mahaleb) regratov med (Taraxacum officinale) cvetlični med Nektar in/ali mana lipov med (Tilia sp.) kostanjev med (Castanea sativa Mill.) javorjev med (Acerpseudoplatanus L., A. platanoides L.) Mana smrekov med (Picea abies (L.) Karst.) hojev med (Abies alba Mill.) gozdni med Po podatkih Statističnega urada RS je v Sloveniji povprečna količina proizvedenega medu 1.800 ton letno. Zaradi dolge tradicije čebelarstva v Sloveniji se večina slovenskega medu porabi doma, delež uvoženega medu predstavlja le 14 %. Po podatkih iz bilance medu se poraba medu v Sloveniji povečuje. Od leta 2000, ko je ocenjena poraba medu na prebivalca znašala nekaj več kot 1 kilogram, se je v zadnjih letih povečala na približno 1,4 kilograma. Povečuje se tudi uvoz medu, saj je domača pridelava medu manjša od skupne porabe. V zadnjih letih uvozimo največ medu iz Hrvaške, Madžarske in Nemčije, izvažamo pa ga v Italijo, Belgijo in Avstrijo (Statistični urad RS, 2019). 2.2 PRIDOBIVANJE CVETNEGA PRAHU OSMU-KANCA Cvetni prah ali pelod je značilen za vsako posamezno cvetočo rastlinsko vrsto. Je osnova spolnega razmnoževanja rastlin, saj vsebuje moške oplojevalne celice rastlin. Čebelji cvetni prah se lahko pridobiva na dva načina, kot izkopanec ali kot osmukanec. Čebele so ana- AAS_vsebina_2020_115_2_ZOOT_110520.indd 224 16. 06. 2020 09:47:59 Pomen čebeljih pridelkov v humani prehrani tomsko prilagojene za nabiranje cvetnega prahu. Pri letu s cveta na cvet se jim cvetni prah oprijema telesa, pokritega z dlačicami, dodajo mu slino in nektar (ali med) iz medenega želodčka ter ga s posebnimi gibi nog zbirajo v koških na zunanji stran nog in prinašajo v panj (Kandolf, 2011). Čebelji prah izkopanec izkopljemo direktno iz čebeljega satja, v katerem je cvetni prah že fermentiran, saj v odsotnosti kisika pride do mlečnokislinskega vrenja. Vendar je to zelo zamudno, količina izkopanca je tudi zelo majhna, zato so oblikovali posebne naprave, osmu-kalnike, s katerimi se pridobiva cvetni prah osmukanec. Osmukalnike namestimo pred vhodom v panj, čebelam pa pri prehodu skozenj iz nožic v zbiralnik odpade nabran cvetni prah (Pucihar, 2017). Cvetni prah osmukanec je svež čebelji pridelek, ki vsebuje veliko vode (20-30 g/100 g), v kombinaciji z visoko hranilno vrednostjo predstavlja idealen vir za razvoj plesni. Zato je potrebno cvetni prah po pobiranju ustrezno predelati in shraniti. Najpogosteje ga stabiliziramo s sušenjem, lahko ga vmešamo tudi v med, zamrznemo ali liofiliziramo (Potokar, 2010). Kakovost cvetnega prahu najbolj ohranimo, če ga svežega nemudoma shranimo v zmrzovalnik (-18 °C), na ta način uničimo tudi morebitne prisotne insekte in mikroorganizme. Po odtalitvi cvetnega prahu je pomembno, da ga takoj porabimo ali ga posušimo v električnih sušilnikih, kjer vlaga enakomerno izhlapeva in temperatura ne presega 40 °C, da preprečimo izgubo vitaminov ter hlapnih snovi, ki prispevajo k oblikovanju arome. Cvetni prah je stabilen, ko vsebuje okoli 4-8 g vode/100 g. Manjša vsebnost vode ni priporočljiva, saj postane senzorično manj sprejemljiv ter težje prebavljiv (Campos in sod., 2008; Bogdanov, 2011). Posušen cvetni prah se lahko skladišči na sobni temperaturi tudi do enega leta in pol, brez spremembe senzorič-nih in mikrobioloških lastnosti. Za ohranitev bioaktivnih spojin je priporočljivo skladiščenje v temnem in suhem prostoru, pri nižjih temperaturah (Bogdanov, 2011). 2.3 PRIDOBIVANJE MATIČNEGA MLEČKA Matični mleček je izloček krmilnih in mandibular-nih žlez mladih čebel delavk (čebel dojilj). Z matičnim mlečkom hranijo vse čebelje ličinke tri dni, po tretjem dnevu pa samo ličinko, iz katere se bo razvila matica. Matica se celo življenje prehranjuje samo z matičnim mlečkom. Matični mleček pridobivajo tako, da čebelji družini brez matice dodajo umetne letvice z matičnimi nastavki, v katere cepijo en dan stare čebelje ličinke, z namenom da jih čebele dojilje preskrbijo z matičnim mlečkom. Večina proizvajalcev matičnega mlečka le-tega pobere tri dni po cepitvi ličink, ker je količina proizvedenega matičnega mlečka takrat največja (Zheng in sod., 2010; Bogdanov, 2011). 2.4 PRIDOBIVANJE PROPOLISA Propolis sestavljajo različne rastlinske smole, ki jih čebele nabirajo na smolnatih popkih dreves, predvsem topola in breze. Dodajo še izločke svojih žlez slinavk in vosek, da postane propolis bolj lepljiv (Huang in sod., 2014; Pasupuleti in sod., 2017). V čebelji družini ima propolis pomembno zaščitno vlogo, čebele ga uporabljajo za premaz svojega bivališča, za zadelovanje notranjih špranj in razpok ter za utrjevanje satja. Čebele s propo-lisom zavarujejo svoje bivališče pred mikroorganizmi in mumificirajo večje tujke v čebelji družini, ki jih fizično ne morejo odstraniti iz panja. Pridobivanje tega čebeljega pridelka je zelo zahtevno, z uporabo posebnih namenskih mrežic iz živilsko neoporečnih materialov, ki jih vstavimo v panj. Te morajo biti mehansko odporne, zdržati morajo različna upogibanja in trenja, tudi po zamrznitvi. Ko mrežice odstranimo iz panjev, jih zavijemo v živilsko folijo, zamrznemo ter nato z njih postrgamo propolis. Iz njega odstranimo vosek, delce čebel in lesene delce. Iz tako pridobljenega propolisa običajno pripravimo etanolno tinkturo (Samec, 2013). 3 sestava čebeljih pridelkov 3.1 SESTAVA MEDU Med je kompleksna naravna mešanica ogljikovih hidratov in drugih spojin, glede na rezultate znanstvenih raziskav vsebuje preko 200 fitokemijskih spojin. Na njegovo sestavo vplivajo različni dejavniki, kot so botanično in geografsko poreklo, klimatske razmere, postopki čebelarjenja, ravnanje z medom in tudi pogoji skladiščenja. Posledica vseh teh dejavnikov je velika raznolikost vrst medu na tržišču (Bogdanov in sod., 2008; Korošec in sod., 2016; Bobi§ in sod., 2017). Med je lahko tekoč ali kristaliziran, odvisno od vsebnosti vode in razmerja med glavnima sladkorjema v medu, fruktozo in glukozo. Med vsebuje tudi druge ogljikove hidrate, di- in tri-saharide. Poleg tega pa so v medu tudi beljakovine, proste aminokisline, organske kisline, fenolne spojine (fenolne kisline in flavonoidi), različni encimi, vitamini in tudi mnogi minerali. Vsebnost glavnih komponent (ogljikovih hidratov) in minornih komponent, pelodnih zrn, aktivnost encimov, vsebnost bioaktivnih spojin (flavonoidov in fenolnih kislin) in senzorične lastnosti medu vplivajo na kakovost in funkcionalne lastnosti tega čebeljega pridelka (Bogdanov, 2008; Viuda-Martos in sod., 2008; Alva- Acta agriculturae Slovenica, 115/2 - 2020 225 AAS_vsebina_2020_115_2_ZOOT_110520.indd 225 16. 06. 2020 09:47:59 M. KOROŠEC in J. BERTONCELJ Preglednica 2: Osnovna sestava različnih vrst slovenskega medu Table 2: The basic composition of Slovenian honey types Sladkorji Voda Fruktoza Glukoza Saharoza Beljakovine Prolin Vrsta medu (g/l00 g) (g/l00 g) (g/l00 g) (g/l00 g) (g/l00 g) (mg/kg) akacijev 13,5-17,5 33,6-45,1 21,9-31,3 2,12-8,28 0,13-0,21 197-447 lipov 14,5-17,8 33,0-43,0 29,5-39,3 0,09-3,51 0,13-0,24 225-398 kostanjev 13,7-18,2 27,7-44,9 17,4-32,7 2,02-3,29 0,31-0,40 457-776 hojev 13,8-17,7 28,1-35,0 23,6-29,6 1,00-4,89 0,18-0,36 323-506 smrekov 14,3-18,5 28,1-42,8 23,1-30,9 1,23-3,75 0,18-0,38 231-495 cvetlični 14,4-18,0 33,2-39,2 28,5-35,5 1,32-4,35 0,18-0,42 309-534 gozdni 13,5-17,0 24,9-36,4 22,9-31,6 1,72-4,61 0,20-0,49 322-461 rez-Suarez in sod., 2009; Yucel in sod., 2017; Korošec in sod., 2017; Combarros-Fuertes in sod., 2019). Osnovna sestava različnih vrst slovenskega medu je podana v preglednici 2. Vsebnost vode je eden najpomembnejših parametrov kakovosti medu, je zakonsko omejena, in sicer je v medu lahko največ 20 g/100 g vode (Pravilnik o medu, 2011). Običajno vsebnost vode v medu ni problematična, v večini vzorcev slovenskega medu se giblje med 14 in 18 g/100 g. Med z večjim odstotkom vode je bolj tekoč in manj viskozen. Majhna vsebnost vode onemogoča rast ozmofilnih kvasovk in tako preprečuje morebitno fermentacijo medu. Glavna sestavina medu so ogljikovi hidrati, ki zajemajo okoli 80 % delež, oziroma okrog 95 % suhe snovi v medu. Količina in razmerje med različnimi ogljikovimi hidrati v medu sta odvisna predvsem od botaničnega porekla, encimov, sestave in intenzivnosti izločanja nektarja, klimatskih razmer ter fiziološkega stanja in moči čebelje družine. Sestava ogljikovih hidratov vpliva na fizikalnoke-mijske lastnosti, kot so viskoznost, kristalizacija in higro-skopnost. Od sladkorjev prevladujeta glukoza in frukto-za, predstavljata od 65 do 90 % vseh ogljikovih hidratov v medu. Kot je razvidno iz preglednice 2, slovenski medovi vsebujejo od 24,9 do 45,1 g fruktoze/100 g in od 17,4 do 39,3 g glukoze/100 g (Korošec in sod., 2016). Podobne vrednosti navajajo za različne vrste medov tudi drugi avtorji (Bogdanov, 2008; Viuda-Martos in sod., 2008; Ajibola in sod., 2012). Disaharidi in oligosaharidi (tri-saharidi) so v medu prisotni v precej manjših količinah, vendar je njihova vsebnost lahko kriterij za določanje botaničnega porekla in pristnosti medu. Saharoze sme biti do 5 g/100 g medu, oziroma v primeru nekaterih izjem, kot je akacijev med, do 10 g/100 g medu (Pravilnik o medu, 2011). Glede na izvor medu, medovi iz nektarja običajno vsebujejo več monosaharidov. Pravilnik o medu (2011) za medove iz nektarja navaja skupno vsebnost fruktoze in glukoze vsaj 60 g/100 g ter za medove iz mane vsaj 45 g/100 g. Medovi iz nektarja in medovi iz mane se običajno ne razlikujejo veliko v vsebnosti disaharidov, obstajajo pa razlike v vsebnosti nekaterih trisaharidov. Er-lozo, maltotriozo in panozo vsebujejo tako nektarne kot manine vrste medu, medtem ko sta rafinoza in melecito-za značilni za manin med (preglednica 3). Prisotnost me-lecitoze v medu iz nektarja tako nakazuje, da je v medu prisotna tudi mana (Korošec in sod., 2016). Preglednica 3: Vsebnost ogljikovih hidratov v slovenskem medu glede na izvor medu Table 3: Carbohydrate composition of Slovenian nectar and honeydew honey types Povprečje ± SD (g/100 g) Ogljikovi hidrati med iz nektarja med iz mane Monosaharidi glukoza 29,38 ± 3,97 26,97 ± 2,43 fruktoza 37,27 ± 2,73 33,31 ± 3,64 Disaharidi saharoza 3,47 ± 1,50 3,03 ± 0,88 maltoza 2,11 ± 0,44 2,07 ± 0,67 palatinoza 0,89 ± 0,08 0,97 ± 0,23 turanoza 1,62 ± 0,25 1,83 ± 0,41 melibioza z gentiobiozo 1,86 ± 0,67 1,83 ± 0,54 Oligosaharidi (trisaharidi) panoza 0,59 ± 0,04 0,61 ± 0,06 erloza 1,60 ± 0,50 2,19 ± 1,03 rafinoza < LOQ 2,21 ± 1,45 melecitoza < LOQ 2,53 ± 1,60 maltotrioza 0,70 ± 0,14 0,92 ± 0,36 SD: standardni odklon; < LOQ: pod mejo kvantitativne določitve 226 Acta agriculturae Slovenica, 115/2 - 2020 AAS_vsebina_2020_115_2_ZOOT_110520.indd 226 16. 06. 2020 09:47:59 Pomen čebeljih pridelkov v humani prehrani Preglednica 4: Vsebnost skupnih fenolnih spojin in antioksi-dativna učinkovitost slovenskega medu Table 4: Total phenolic content and antioxidant activity of Slovenian honey types Vrsta medu Vsebnost skupnih fenolnih spojin (mg GAE/l00 g) Antioksidativna učinkovitost (FRAP) (pM Fe(II)) akacijev 25,7- 67,9 56,8- -86,0 lipov 63,4- 109,0 94,6- 155,1 kostanjev 146,8- 272,3 238,3- 469,5 hojev 163,4- 285,7 320,8- 582,2 smrekov 185,7- 239,0 277,5- 495,4 cvetlični 126,8- 194,6 181,1- 262,9 gozdni 192,3- -270,1 371,6- -494,1 GAE: ekvivalent galne kisline; FRAP: ferric reducing antioksidant power (antioksidativna moč redukcije železa) Poleg ogljikovih hidratov vsebuje med številne organske in tudi anorganske kisline, katerih skupno vsebnost izražamo v miliekvivalentih. Prostih kislin sme med vsebovati do 50 mekv/kg (Pravilnik o medu, 2011). Beljakovine v medu nimajo velikega prehranskega pomena, saj je njihova vsebnost majhna, običajno pod 0,5 g/100 g. V primeru slovenskega medu največ beljakovin v povprečju vsebuje kostanjev med, najmanj pa aka-cijev med (preglednica 2). Aminokisline v medu izvirajo iz nektarja oz. mane, cvetnega prahu in čebel. Njihova vsebnost v medu je zelo majhna, največ je prolina, ki je v povezavi z zrelostjo medu, botaničnim poreklom in pristnostjo (Korošec in sod., 2017). Med je tudi naravni vir antioksidantov, med katerimi so najbolj pomembne fenolne kisline, flavonoi-di, encimi (glukoza oksidaza, katalaza, peroksidaza) in produkti Maillardove reakcije (Bertoncelj in sod., 2007; Bogdanov, 2008; Ajibola, 2015; Bobi§ in sod., 2017; Pasu-puleti in sod., 2017). Na vsebnost fenolnih spojin v medu vplivajo botanično in geografsko poreklo medu ter podnebne razmere. V medu so od flavonoidov prisotni predvsem flavoni, flavonoli in flavanoni ter različne fenolne kisline (Viuda--Martos in sod., 2008; Bertoncelj in sod., 2011). Skupna vsebnost fenolnih spojin v slovenskih medovih je podana v preglednici 4, kjer so razvidne velike razlike med posameznimi vrstami medu, najmanj jih vsebuje akacijev med, največ pa medovi iz mane, hojev, gozdni in smrekov med (Bertoncelj in sod., 2007). Vsebnost fenolnih spojin je v tesni povezavi z an-tioksidativno učinkovitostjo. Medovi z večjo vsebnostjo fenolnih spojin, imajo večjo antioksidativno učinkovitost, določeno s FRAP metodo (Bertoncelj in sod., 2007; Korošec in sod., 2017). Vsebnost skupnega pepela v medu je količina anorganskega ostanka po sežigu medu in ponazarja količino v medu prisotnih mineralnih snovi. Določanje pepela je dokaj zahtevno, zato se nadomešča z merjenjem električne prevodnosti medu, saj med tema dvema parametroma obstaja linearna zveza. Čim več je v medu prisotnih mineralnih snovi, večja je vsebnost skupnega pepela in višja je električna prevodnost (Kropf in sod., 2008). Med kot živilo ni pomemben vir elementov, skupna vsebnost pepela v medu iz nektarja običajno znaša pod 0,6 g/100 g, v medu iz mane pa do 1,0 g/100 g (preglednica 5). Raznolikost elementov v posameznem vzorcu medu je v veliki meri odvisna od sestave nektarja, mane in prsti ter prevladujočega cvetnega prahu. Iz skupine makroe-lementov v medu je samo kalij prisoten v količinah nad 200 mg/kg. Med vsebuje tudi različne mikroelemente (vsebnost nad 1 mg/kg) ter elemente v sledovih. Različne študije so pokazale, da ima botanično poreklo največji vpliv na vsebnost elementov v sledovih v medu (Korošec in sod., 2017). Preglednica 5: Vsebnost pepela in nekaterih elementov v različnih vrstah slovenskega medu Table 5: Ash and elemental content in different Slovenian honey types Pene! Povprečna vsebnost elementov ± SD (mg/kg) Vrsta medu (g/kg) K Cl Ca S Rb Mn Br akacijev 0,4-0,9 278 ± 78 95 ± 52 17,3 ± 7,7 47 ± 19 0,72 ± 0,32 1,68 ± 1,27 0,60 ± 0,26 lipov 1,8-3,0 1800 ± 349 379 ± 139 69 ± 23 50 ± 27 5,5 ± 2,9 3,55 ± 1,56 1,02 ± 0,43 kostanjev 5,5-10,4 3590 ± 657 240 ±217 148 ± 33 42 ± 24 17,0 ± 7,7 23,2 ± 9,0 0,55 ± 0,23 hojev 3,8-7,1 3170 ± 555 333 ± 134 35 ± 18 71 ± 26 22,0 ± 7,0 5,03 ± 1,93 0,59 ± 0,12 smrekov 4,1-6,5 2950 ± 494 322 ± 74 47 ± 17 70 ± 26 13,9 ± 6,1 7,07 ± 2,3 0,58 ± 0,22 cvetlični 1,1-2,7 1120 ± 352 264 ± 85 61 ± 25 56 ± 25 2,97 ± 1,63 3,12 ± 1,59 0,65 ± 0,25 gozdni 4,4-6,3 2940 ± 561 310 ± 79 59 ± 19 57 ± 21 13,7 ± 7,8 6,74 ± 2,51 0,59 ± 0,25 SD: standardni odklon Acta agriculturae Slovenica, 115/2 - 2020 227 AAS_vsebina_2020_115_2_ZOOT_110520.indd 227 16. 06. 2020 09:47:59 M. KOROŠEC in J. BERTONCELJ Preglednica 6: Osnovna hranilna sestava mešanega cvetnega prahu osmukanca slovenskega izvora Table 6: Basic nutritional composition of Slovenian bee pollen Vsebnost v svežem vzorcu Vsebnost na suho snov Parameter povprečje x . min x max povprečje x min x max voda (g/100 g) 22,73 15,70 29,20 beljakovine (g/100 g) 17,46 13,00 22,90 22,73 16,03 32,34 maščobe (g/100 g) 7,36 4,50 12,30 9,55 6,07 15,79 pepel (g/100 g) 2,06 1,30 2,80 2,67 1,65 3,88 skupni ogljikovi hidrati (g/100 g) 50,4 39,3 60,0 65,05 54,75 73,98 energijska vrednost (kJ/100 g) 1430 1300 1540 1850 1780 1980 x . : minimalna vrednost, x : maksimalna vrednost 3.2 SESTAVA CVETNEGA PRAHU Cvetni prah osmukanec vsebuje enostavne sladkorje, vse esencialne aminokisline, nasičene in nenasičene maščobne kisline, nekatere elemente (K, Mg, Zn, Cu, Fe) in vitamine (vitamini skupine B, ß-karoten, vitamin E, vitamin C), sekundarne rastlinske metabolite (flavonoidi, fitosteroli) ter prehransko vlaknino. Zaradi močne raznolikosti in prisotnih zrn cvetnega prahu različnih rastlin je v mešanem cvetnem prahu opazen velik razpon med najnižjo in najvišjo vrednostjo za vsebnost posamezne hranljive snovi (Campos in sod., 2008; Campos in sod., 2016). Cvetni prah predstavlja tudi odličen vir energije, energijska vrednost variira med 1590 in 2050 kJ/100 g (Yang in sod., 2013; Bogdanov, 2016). Cvetni prah vsebuje veliko različnih biološko aktivnih spojin, kot so flavonoidi (katehin, kamferol, kver-cetin, izoramnetin), fitosteroli in karotenoidni pigmenti (likopen in zeaksantin), ki lahko delujejo antioksidativ-no, protimikrobno, antikancerogeno in protivnetno (Ko-mosinska-Vassev in sod., 2015; Denisow in Denisow-Pi-etrzyk, 2016; Bogdanov, 2016; Kaškoniene in sod., 2020). Iz preglednice 6, kjer je podana hranilna vrednost slovenskega cvetnega prahu osmukanca (Lilek in sod., 2015), je razvidno, da od hranljivih snovi cvetni prah vsebuje največ ogljikovih hidratov, sledijo beljakovine in maščobe. Ker je vsebnost vode v svežem cvetnem prahu osmukancu zelo variabilna, so rezultati podani tudi na suho snov. Podrobnejša analiza ogljikohidratne sestave cvetnega prahu osmukanca (Pucihar, 2017; Bertoncelj in sod., 2018) je pokazala, da med enostavnimi ogljikovimi hidrati v cvetnem prahu prevladujeta fruktoza (od 13,17 do 27,84 g/100 g suhe snovi) in glukoza (od 10,60 do 28,49 g/100 g suhe snovi). Cvetni prah pa je tudi dober vir prehranske vlaknine, vsebnost topne prehranske vlaknine je v območju od 0,62 do 5,21 g/100 g suhe snovi, vsebnost netopne prehranske vlaknine pa od 7,72 do 17,89 g/100 g suhe snovi (Bertoncelj in sod., 2018). Cvetni prah vsebuje tudi različne elemente in vitamine (preglednica 7). Variabilnost v njihovi vsebnosti, predvsem pri mešanih vrstah cvetnega prahu, pripisujejo različnim vrstam cvetnega prahu rastlin. Od elementov je najbolj zastopan kalij (60 % od skupne vsebnosti) Preglednica 7: Vsebnost elementov in vitaminov v cvetnem prahu osmukancu (Campos in sod., 2008) Table 7: Contents of elements and vitamins in bee pollen (Campos et al., 2008) Elementi mg/100 g suhe snovi Vitamini mg/100 g suhe snovi kalij (K) 400-2000 provitamin A (p-karoten) 1-20 magnezij (Mg) 20-300 vitamin B1 (tiamin) 0,6-1,3 kalcij (Ca) 20-300 vitamin B2 (riboflavin) 0,6-2 fosfor (P) 80-600 vitamin B3 (niacin) 4-14 železo (Fe) 1,1-17 vitamin B5 (pantotenska kislina) 0,5-2 cink (Zn) 3-25 vitamin B6 (piridoksin) 0,2-0,7 baker (Cu) 0,2-1,6 vitamin B7 (biotin) 0,05-0,07 mangan (Mn) 2-11 vitamin B9 (folna kislina) 0,3-1 vitamin C (askorbinska kislina) 7-56 vitamin E (tokoferol) 4-32 228 Acta agriculturae Slovenica, 115/2 - 2020 AAS_vsebina_2020_115_2_ZOOT_110520.indd 228 16. 06. 2020 09:47:59 Pomen čebeljih pridelkov v humani prehrani (Campos in sod., 2008). Cvetni prah je dober vir vitaminov, topnih v vodi, vitaminov skupine B ter vitamina C (Soares de Arruda in sod., 2013). 3.3 SESTAVA MATIČNEGA MLEČKA Sestava matičnega mlečka je kompleksna, odvisna je od sezonskih in okoljskih dejavnikov, načina pridobivanja ter prehrane in starosti čebel. Vsebnost vode je v matičnem mlečku zelo visoka (od 60 do 70 %), suho snov pa predstavljajo ogljikovi hidrati, proteini, bioaktivni pepti-di, aminokisline, maščobe ter manjša količina vitaminov in mineralov (Sabatini in sod., 2009). Matični mleček vsebuje večinoma zelo specifične kratkoverižne mono- in dihidroksi maščobne kisline z 8-10 ogljikovimi atomi ali dikarboksilne kisline. Za matični mleček je specifična trans-10-hidroksi-2-decenoj-ska kislina (10-HDA), ki jo je v matičnem mlečku največ (več kot 50 % vseh maščobnih kislin). 10-HDA je značilna samo za matični mleček, zato je njena vsebnost v matičnem mlečku pomemben kriterij njegove pristnosti (Ramadan in Al-Ghamdi, 2012). Sestava sladkorjev, vsebnost vode, beljakovin in 10-HDA so najbolj pomembni kriteriji za karakterizacijo matičnega mlečka (Sabatini in sod., 2009; Bobi§ in sod., 2017). Pomemben kriterij kakovosti matičnega mlečka so tudi senzorične lastnosti. Matični mleček je umazano bele do bledo rumene barve, gosto tekoč, pogosto nehomogen (zrnast, peskast) zaradi prisotnosti netopnih granul različnih velikosti. Ima vonj po kislem, rezkem, kisel okus ter ostro, pikantno aromo, lahko po vosku, po živalih. S staranjem barva matičnega mlečka temni, okus lahko postane žarek (ISO, 2016). V preglednici 8 je podan predlog standardne sestave matičnega mlečka in rezultati analiz slovenskega matičnega mlečka. 3.4 SESTAVA PROPOLISA Sestava propolisa je zelo raznolika, odvisna je od rastlin, na katerih so čebele nabirale surovino, od klimatskih razmer v času nabiranja pa tudi od načina pridobivanja in vrste čebel, ki imajo različne preference do posameznih rastlin. Propolis vsebuje različne smole (50 %), voske (30 %), eterična olja in druge aromatične spojine (10 %), cvetni prah (5 %) ter druge sestavine, kot so aminokisline, vitamini in minerali (Viuda-Martos in sod., 2008; Pasupuleti in sod., 2017). V propolisu so identificirali več sto različnih spojin. Glavne so terpenoidi in fenolne spojine, kamor spadajo flavonoidi ter fenolne kisline in njihovi estri, ki so odgovorni za protivirusno in protivnetno delovanje propolisa. Naravne fenolne spojine delujejo tudi kot antioksidanti. Najbolj značilne fenolne spojine propolisa so flavonoidi pinocembrin, pinobanksin, krizin, galangin, kamferol in kvercetin, fenolne kisline cimetna, p-kumarna, kavna in ferulna kislina ter fenetilni ester kavne kisline (CAPE) in artepilin C (Huang in sod., 2014). 4 uporaba čebeljih pridelkov v prehrani Med, cvetni prah, matični mleček in propolis so čebelji pridelki, ki jih ljudje uživajo zaradi odlične hranilne vrednosti, kot tudi zaradi njihovih funkcionalnih lastnosti in biološke aktivnosti. Zaradi svojih lastnosti so tudi primeren dodatek oz. potencialna sestavina za različna živila. Potrebno pa je upoštevati nekatere previdnostne ukrepe za uporabo v prehrani v samostojni obliki ali kot dodatek živilom, da bi se izognili morebitnim alergijskim reakcijam pri osebah, občutljivih na posamezne čebelje pridelke oz. katero od njihovih sestavin. Zato je potrebno Preglednica 8: Sestava svežega matičnega mlečka Table 8: Composition of fresh royal jelly Parameter Predlog standardne sestave1 Zahtevana sestava po ISO 128242 Slovenski matični mleček 3, 4 vsebnost vode (g/100 g) 60-70 62,0-68,5 62,0-66,7 vsebnost maščob (g/100 g) 3-8 2-8 4,44-6,19 vsebnost 10-HDA (g/100 g) > 1,4 > 1,4 2,32-3,21 vsebnost beljakovin (g/100 g) 9-18 11-18 11,6-13,6 vsebnost fruktoze (g/100 g) 3-13 2-9 2,3-4,5 vsebnost glukoze (g/100 g) 4-8 2-9 3,4-5,3 vsebnost saharoze (g/100 g) 0,5-2,0 < 3,0 0-2,0 vsebnost pepela (g/100 g) 0,8-3,0 / 0,94-1,23 1 Sabatini in sod., 2009, 2 ISO 12824: 2016; 3 Štaudohar, 2014; 4 Kandolf Borovšak in sod., 2017; / ni podatka Acta agriculturae Slovenica, 115/2 - 2020 229 AAS_vsebina_2020_115_2_ZOOT_110520.indd 229 16. 06. 2020 09:47:59 M. KOROŠEC in J. BERTONCELJ z uživanjem čebeljih pridelkov začeti previdno in zaužito količino povečevati postopoma (Bogdanov, 2011). Med ljudje v vsakdanji prehrani uživamo že od nekdaj, v zadnjem času narašča uporaba cvetnega prahu, matični mleček in propolis pa se uživata predvsem v obliki prehranskih dopolnil in uporabljata v apiterapiji. Apite-rapija je veda o tem, kako si s pomočjo čebeljih pridelkov krepimo in ohranjamo zdravje. Začetki apiterapije segajo stoletja nazaj do egipčanske, grške, kitajske, babilonske in drugih civilizacij. Trditve o zdravilnih učinkih apiterapije temeljijo predvsem na dejanskih izkušnjah posameznikov in tradicionalni uporabi (Bogdanov, 2011; Fratellone in sod., 2016, Yucel in sod., 2017). Med je eno najbolj kompleksnih naravnih živil in edino sladilo, ki ga človek uporablja brez predhodne predelave. Uživanje medu je primerno za ljudi vseh starostnih skupin, tudi za nosečnice in doječe matere, le dojenčkom in otrokom do 1. leta starosti ga zaradi možnosti prisotnih spor Clostridium botulinum ne smemo ponuditi. V okviru zagotavljanja uravnotežene prehrane in skrbi za zdravje je vsekakor priporočljivo, da ga vključimo v vsakodnevne obroke in z njim nadomestimo kuhinjski sladkor in druga sladila. Ogljikovi hidrati so glavna sestavina medu in s pre-hranskega vidika zelo pomembni. Enostavna sladkorja, glukoza in fruktoza, predstavljata hitro izkoristljiv vir energije. Ob zamenjavi kuhinjskega sladkorja z medom pa hkrati vnesemo tudi manjše količine vitaminov, mineralov in drugih bioaktivnih spojin, ki jih sladkor ne vsebuje. Zaradi velike vsebnosti fruktoze ima med manjši vpliv na raven glukoze v krvi kot bel sladkor, kar vpliva tudi na vrednost glikemijskega indeksa. Deibert in sod. (2010) so na osnovi klinične študije z 10 udeleženci, za pet od osmih vrst nemškega medu, določili nizke vrednosti glikemijskega indeksa, pod 55. Samo gozdni med je imel vrednost nad 70, kar predstavlja visok glikemijski indeks. Vrste medu z nizkim glikemijskim indeksom, ki vsebujejo več fruktoze kot glukoze (npr. akacijev in kostanjev med), bi lahko pod ustreznim nadzorom uživali tudi diabetiki oziroma bi se potencialno lahko uporabile za obvladovanje slakorne bolezni. Vendar, ker mehanizem hipoglikemičnega učinka medu ni pojasnjen, velja v praksi zadržanost in previdnost (Meo in sod., 2017; Bobi? in sod., 2018). Rezultati raziskav kažejo, da je za doseganje ugodnih učinkov medu na prehranski status in zdravje posameznika potrebno uživati večje količine medu, od 50 do 80 g (Bogdanov in sod., 2008) oz. od 70 do 90 g (Aji-bola, 2015) dnevno, kar ni v skladu s priporočili za vnos prostih sladkorjev (WHO, 2015), h katerim prištevamo sladkorje iz medu. Med vsebuje tudi več oligosaharidov in nekaj poli-saharidov z nizko molekulsko maso in ima zato prebio- tične lastnosti. Oligosaharidi so ogljikovi hidrati s 3 do 9 monomernimi enotami, ki so rezistentni na prebavo v tankem črevesu človeka, delno se razgradijo v debelem črevesu do kratkoverižnih maščobnih kislin, ki predstavljajo pomembno hranilo za mikrobioto (Mohan in sod., 2017; Cornara in sod., 2017). Oligosaharidi povečajo število in aktivnost koristnih mikroorganizmov (lakto-bacilov in bifidobakterij) v prebavnem traktu (Ajibola, 2015; Begum in sod., 2015; Yucel in sod., 2017; Pasupu-leti, 2017). Medovi iz mane vsebujejo večjo količino (do 10 g/100 g) in več različnih oligosaharidov, zato imajo tudi večji prebiotični učinek (Bogdanov in sod., 2008). Vsebnost posameznih vitaminov in elementov v medu je majhna, zato je njihov prispevek pri priporočenih dnevnih vnosih (Referenčne vrednosti..., 2016) zgolj neznaten. Če izpostavimo kalij, ki ga je v medu največ, bi z eno žlico medu (približno 20 g) zaužili do 5,5 % dnevnega vnosa, odvisno od vrste medu (Kropf in sod., 2009). To pomeni, da med ni pomemben vir kalija v naši prehrani, je pa v oziru zastopanosti elementov ustreznejša prehranska izbira med sladili kot kuhinjski sladkor. Med fiziološke učinke medu spadajo poleg že omenjenega prebiotičnega učinka tudi antioksidativna in protimikrobna učinkovitost, protivnetno delovanje in zaviranje encimskega porjavenja. Medu pripisujejo tudi ugodne učinke pri zdravljenju različnih bolezni, preprečevanju pojava določenih bolezni, tudi rakavih obolenj. Po navedbah mnogih avtorjev vpliva med ugodno na delovanje srca, upočasnjuje razvoj ateroskleroze, pospešuje izločanje strupov iz telesa in pomaga pri boleznih dihal. Vse te funkcionalne lastnosti se večinoma pripisujejo fenolnim spojinam, kot so flavonoidi (Bogdanov in sod., 2008; Viuda-Martos in sod., 2008; Bobi§ in sod., 2017; Pasupuleti in sod., 2017; Combarros-Fuertes in sod., 2019) in bioaktivnim peptidom, kot so defensin-1 in že-leini (Cornara in sod., 2017). Med zavira rast in razvoj velikega števila mikroorganizmov zaradi velike vsebnosti sladkorjev, ki povzročijo osmotski učinek, majhne vsebnosti vode, nizke vrednosti vodne aktivnosti, nizke vrednosti pH in prisotnosti spojin z antimikrobnim delovanjem. Pod vplivom encima glukoza oksidaza, ki ga vsebuje med, nastane vodikov peroksid, ki pripomore k celjenju tkiva in deluje antibakterijsko. Zaradi teh lastnosti med že od nekdaj uporabljajo tudi za zdravljenje ran (Molan, 2006; Ajibola in sod., 2012; Yilmaz in Aygin, 2020), hkrati pa lahko pripomore tudi k zdravju ustne votline, saj preprečuje rast bakterij, ki povzročajo karies, manj vpliva na erozijo zobne sklenine (Bogdanov in sod., 2008) ter je lahko učinkovito sredstvo proti parodontozi, saj zavira delovanje parodontopatogenih bakterij in tako predstavlja cenejšo alternativno metodo zdravljenja (Po-držaj, 2011). Čebelji cvetni prah, kamor prištevamo cvetni prah 230 Acta agriculturae Slovenica, 115/2 - 2020 AAS_vsebina_2020_115_2_ZOOT_110520.indd 230 16. 06. 2020 09:47:59 Pomen čebeljih pridelkov v humani prehrani osmukanec in cvetni prah izkopanec, je pomemben vir hranil in energije in lahko predstavlja dodatek k vsakodnevni prehrani. Cvetni prah osmukanec vsebuje vse potrebne esencialne spojine, potrebne v prehrani človeka, vključno z aminokislinami in maščobnimi kislinami. Bogdanov (2011) navaja, da je dnevni vnos 10 g cvetnega prahu realen glede na ceno tega čebeljega pridelka in že omogoča preventivno delovanje. Za preventivo in izboljšanje zdravja se priporoča od 10 do 20 g cvetnega prahu dnevno, običajno 3 mesece zaporedoma, 2-krat letno. V apiterapiji pa je dnevni odmerek cvetnega prahu večji, od 20 do 50 g dnevno, zaužit 3-krat dnevno, 1 do 2 uri pred obrokom. Za večjo senzorično sprejemljivost cvetnega prahu se priporoča mešanje z medom, s sokom ali z mlečnimi izdelki, npr. jogurti ali s skuto ter sadjem. Za povečanje prebavljivosti cvetnega prahu osmukanca v organizmu in s tem razpoložljivosti posameznih hranil je ta čebelji izdelek priporočljivo pred uporabo zmleti ali namočiti zrna v topli vodi ali drugi tekočini, s čimer ovojnica zrna cvetnega prahu postane bolj prepustna (Bogdanov, 2011; Komosinska-Vassev in sod., 2015; Denisow in Denisow-Pietrzyk, 2016; Yucel in sod., 2017). Zaradi velike variabilnosti v sestavi cvetnega prahu (preglednici 6 in 7), kot posledici različnega botaničnega izvora, je težko realno oceniti vnos posameznih makro- in mikrohranil v prehrani z uživanjem dnevno priporočene količine cvetnega prahu. Cvetni prah je dober vir beljakovin in esencialnih aminokislin ter maščobnih kislin, sladkorjev fruktoze in glukoze, kot tudi nekaterih vitaminov in mineralov. Doprinos teh komponent k priporočenemu dnevnemu vnosu je pri cvetnem prahu večji kot pri medu. Cvetni prah je tudi dober vir prehranske vlaknine (Bertoncelj in sod., 2018), komponente, za katero je ocenjeni vnos s prehrano pri prebivalcih razvitih držav prenizek glede na priporočen dnevni vnos, ki znaša najmanj 30 g na dan za odrasle (Referenčne vrednosti..., 2016). Zaužitje 20 g cvetnega prahu dnevno bi prispevalo okoli 10 % priporočenega dnevnega vnosa prehranske vlaknine. Na osnovi vsebnosti naštetih hranljivih snovi cvetni prah izboljša presnovo ter splošno telesno zmogljivost in je zelo primeren za okrevanje po boleznih ter za ljudi s premajhno telesno maso. Kot kažejo številne raziskave v zadnjih letih ima cvetni prah poleg visoke hranilne vrednosti tudi veliko vsebnost biološko aktivnih snovi (flavonoidov, fitostero-lov, različnih encimov), ki prispevajo k številnim funkcionalnim lastnostim (Komosinska-Vassev in sod., 2015; Denisow in Denisow-Pietrzyk, 2016; Yucel in sod., 2017; Cornara in sod., 2017; Kaškoniene in sod., 2020). Fla-vonoidi delujejo antioksidativno, protimikrobno, anti-kancerogeno in protivnetno, ščitijo pred pojavom atero-skleroze in drugih bolezni srca in ožilja, krepijo imunski sistem ter zavirajo prehitro staranje. Fitosteroli vplivajo na nivo holesterola v krvi in z njim povezanih bolezni srca in ožilja, zavirali naj bi tudi nastanek nekaterih vrst raka. Vse biološko aktivne spojine v cvetnem prahu imajo močno protivnetno delovanje in spodbujajo delovanje imunskega sistema. Cvetni prah tako nima le vloge pre-hranskega dodatka in funkcionalnega živila, ampak tudi potencialnega zdravila. Natančno stopnjo biološke učinkovitosti pa je težko določiti zaradi velike variabilnosti v sestavi tega čebeljega pridelka, ki je odvisna od botaničnega izvora. Za namen zdravljenja je nujno potrebno definirati standarde kakovosti cvetnega prahu, ki bi olajšali uporabo cvetnega prahu v medicinske namene (Denisow in Denisow-Pietrzyk, 2016). Matični mleček in propolis se bolj kot živilo uporabljata v obliki prehranskih dopolnil ali v kombinaciji z medom. Matični mleček zaradi njegovih specifičnih senzoričnih lastnosti in visoke cene potrošniki bolj dojemajo kot domače zdravilo. Biološka aktivnost matičnega mlečka se pripisuje predvsem maščobnim kislinam (10-HDA), bioaktivnim peptidom, specifičnim prote-inom matičnega mlečka (npr. rojalaktina in rojalizina) in fenolnim spojinam. Matični mleček se že od davnih časov uporablja v tradicionalni medicini, zaradi številnih pozitivnih lastnosti sodi v skupino funkcionalnih živil. Nekateri biološki in terapevtski učinki uživanja matičnega mlečka so že bili potrjeni, vendar pa kemijska sestava in biološko aktivne snovi matičnega mlečka še niso v celoti poznane. Z različnimi raziskavami so dokazali an-tioksidativno, protibakterijsko in protivnetno delovanje matičnega mlečka (Ramadan in Al-Ghamdi, 2012; Pasu-puleti in sod., 2017; Ahmad in sod., 2020). Matični mleček antibakterijsko deluje tako na gram-pozitivne kot na gram-negativne bakterije, učinek pripisujejo specifičnim prostim maščobnim kislinam ter proteinom matičnega mlečka (Ramadan in Al-Ghamdi, 2012). Zelo razširjena je uporaba matičnega mlečka v prehranskih dopolnilih zaradi prepričanja, da ima njegovo uživanje podobne učinke na ljudi, kot jih ima na čebele. Čebela matica, ki je izključno hranjena z matičnim mlečkom, ima daljšo življenjsko dobo ter bolj razvite žleze v primerjavi s čebelo delavko (Morita in sod., 2012). Uživanje matičnega mlečka vpliva tudi na boljšo vzdržljivost pri športnikih. Športniki, ki uživajo matični mleček kot prehransko dopolnilo (1,2 g/dan), so bolj vzdržljivi v primerjavi s športniki, ki tega prehranskega dodatka ne uživajo. Med telesno dejavnostjo vzdržljivost pada zaradi povišanja li-pidnih hidroperoksidov v krvi. Matični mleček s svojimi sestavinami kot antioksidant pomaga pri zniževanju hi-droperoksidov v krvi in posledično pripomore k boljši vzdržljivosti (Bogdanov, 2011). Matični mleček zaradi vsebnosti specifičnih proteinov in fenolnih spojin, ki so zelo učinkovite pri odstranjevanju prostih radikalov, deluje tudi antioksidativno (Bobi§ in sod., 2017: Cornara in Acta agriculturae Slovenica, 115/2 - 2020 231 AAS_vsebina_2020_115_2_ZOOT_110520.indd 231 16. 06. 2020 09:47:59 M. KOROŠEC in J. BERTONCELJ sod., 2017; Yucel in sod., 2017). Študije na živalih kažejo, da ima matični mleček tudi antitumorsko delovanje, ki se ga pripisuje predvsem vsebnosti 10-HDA ter nasičenim dikarboksilnim kislinam (Oršolic, 2013). Tudi propolis je del tradicionalne medicine, vsebuje številne bioaktivne spojine, zlasti fenolne spojine, ki delujejo antioksidativno, protivirusno in protivnetno (Huang in sod., 2014; Cornara in sod., 2017; Pasupuleti in sod., 2017). 5 pomen standardizacije čebeljih pridelkov Na osnovi različnih bioloških lastnosti čebeljih izdelkov, dokazanih z znanstvenimi študijami, so bili izvedeni tudi poskusi aplikacij nekaterih od teh pridelkov v kliničnih okoljih, vendar je njihova farmakološka in medicinska standardizacija zaradi velike kemijske variabilnosti otežena, biološka učinkovitost čebeljih pridelkov je namreč odvisna od botaničnega in geografskega porekla, vrste medonosnih čebel, postopkov čebelarjenja in postopkov s pridelki po njihovem pridobivanju. Izolirane so bile različne spojine z dokazanim biološkim učinkom, kar kaže na pomembnost čebeljih pridelkov za odkrivanje zdravil iz naravnih virov (Cornara in sod., 2017; Pasupuleti in sod., 2017; Ahmad in sod., 2020). Potrebne pa so dodatne, ustrezne klinične študije za potrditev aktivnosti čebeljih pridelkov oz. njihovih sestavin. Zaradi nezadostnih utemeljenih znanstvenih dokazov o učinkovanju na zdravje do sedaj tudi ni bila odobrena nobena zdravstvena trditev za čebelje pridelke. Zdravstvena trditev pomeni vsako trditev, ki navaja, domneva ali namiguje, da obstaja povezava med kategorijo živil, živilom ali eno od njegovih sestavin na eni strani in zdravjem na drugi strani (Uredba 1924/2006). V postopku sprejemanja novih zdravstvenih trditev za živila je bilo na Evropsko agencijo za varnost hrane (EFSA) vloženih tudi nekaj vlog zdravstvenih trditev za matični mleček in propolis (Vujič in Pollak, 2015). Predlagane trditve so se nanašale na krepitev imunskega sistema, vitalnost organizma, povečanje antioksidativne sposobnosti organizma, ohranjanje zdravega delovanja jeter, povečanje fiziološke odpornosti organizma, krepitev zdravja zgornjih dihal, izboljšanje kakovosti življenja žensk v menopavzi, spodbujanje delovanja srca, uravnotežen nivo lipidov v krvi. EFSA je pri presojanju upravičenosti trditev za tradicionalna živila, ki se uporabljajo za domače zdravljenje, zaradi njihove naravne in sezonske variabilnosti precej zadržana. Zato je vse predlagane zdravstvene trditve zavrnila, ker živilo oziroma sestavina živila ni bila dovolj dobro opredeljena in karakterizirana ter za zatrjevani učinek ni bilo dovolj utemeljenih znanstvenih dokazov, tudi zaradi pomanjka- 232 Acta agriculturae Slovenica, 115/2 - 2020 nja ustreznih kliničnih študij. Seznam zavrnjenih zdravstvenih trditev za čebelje pridelke in utemeljitve njihove zavrnitve so dostopni na spletni strani Evropske komisije, ki vodi tako imenovani Register skupnosti v zvezi s prehranskimi in zdravstvenimi trditvami (European Commission, 2020). Standardizacija posameznih čebeljih pridelkov v smislu standardizacije njihove sestave, ki vključuje tako vsebnost makro- in mikrohranil, kot tudi bioaktivnih spojin, je pomembna tudi za lažje vrednotenje doseganja priporočenih vrednosti posameznih hranljivih snovi glede na priporočila za vnos energije in hranil (Referenčne vrednosti..., 2016). Na ta način bi lahko v prehrano vključili čebelje pridelke z optimalnimi lastnostmi za potrebe posameznika. Hkrati pa bi standardizacija omogočila lažje preverjanje pristnosti čebeljih pridelkov, ker so le-ti zaradi visoke cene podvrženi tudi potvorbam, z definirano sestavo pa bi potvorjene čebelje pridelke lažje odkrili in s tem tudi zaščitili potrošnika. 6 dodatek čebeljih pridelkov drugim živilom V današnjem času potrošniki želijo živila, ki so bolj naravna in vsebujejo manj aditivov. Med in ostali čebelji pridelki kažejo nekatere pozitivne lastnosti, ki omogočajo njihov dodatek v različna živila. Med lahko nadomesti nekatere konvencionalne dodatke, kar omogoča tudi razvoj novih živil. Med se že od nekdaj uporablja kot sla-dilo v različnih pijačah in živilih, kot so brezalkoholne sadne pijače, jogurtovi napitki, športni napitki. V mleku in mlečnih izdelkih med spodbuja rast mlečnih starter kultur zaradi prisotnih oligosaharidov. Med tudi preprečuje encimsko porjavenje sadja in zelenjave in izdelkov iz njih in bi se lahko uporabljal kot alternativa sulfitom. V pekovskih izdelkih dodatek medu vpliva na zadrževanje vode, kar povzroči boljšo teksturo in izboljšane ostale senzorične lastnosti. V mesu in mesnih izdelkih med lahko preprečuje mikrobiološki kvar in oksidacijo maščob ter zmanjša nastanek heterocikličnih aromatskih aminov (Viuda-Martos in sod., 2008; Bogdanov, 2011; Yucel in sod., 2017). Cvetni prah osmukanec se dodaja predvsem mlečnim in pekovskim izdelkom za izboljšanje njihove pre-hranske vrednosti. Atallah (2016) navaja, da so imeli probiotični jogurti z dodatkom cvetnega prahu (0,8 %) ali matičnega mlečka (0,6 %) boljše senzorične lastnosti (aromo, teksturo in celokupno všečnost), večjo vsebnost nekaterih elementov (Ca, P, K, Mg, Mn, Fe in Zn) ter boljše reološke lastnosti (manjša sinereza) v primerjavi z jogurtom brez dodatka prej omenjenih čebeljih pridelkov. Krystyjan in sod. (2015) so proučevali fizikalno- AAS_vsebina_2020_115_2_ZOOT_110520.indd 232 16. 06. 2020 09:47:59 Pomen čebeljih pridelkov v humani prehrani kemijske in antioksidativne lastnosti keksov z dodanim cvetnim prahom. Pri pripravi keksov so del moke (od 2,5 do 10 %) nadomestili z mletim cvetnim prahom. Dodatek cvetnega prahu je vplival na povečanje vsebnosti beljakovin, sladkorjev, prehranske vlaknine in fenolnih spojin ter večjo antioksidativno učinkovitost in izboljšane senzorične lastnosti obogatenih keksov. Podobno Sol-gajova in sod. (2014) navajajo, da imajo keksi, obogateni s cvetnim prahom oljne ogrščice, boljšo tako hranilno vrednost kot tudi tehnološke in senzorične lastnosti. Matični mleček in propolis se običajno dodajata medu za povečanje vsebnosti bioaktivnih učinkovin in večjo antioksidativno učinkovitost (Juszczak in sod., 2016). 7 zaključek Čebelji pridelki so popolnoma naravna živila, ki jih lahko uživamo samostojno ali kot dodatek drugim živilom za izboljšanje njihove hranilne vrednosti in funkcionalnih lastnosti. Med, cvetni prah osmukanec in matični mleček imajo visoko hranilno vrednost in dokazano biološko delovanje. Z uživanjem čebeljih pridelkov dopri-nesemo k vnosu hranljivih snovi in dodatno zagotovimo organizmu tudi bioaktivne spojine, ki lahko ugodno vplivajo na zdravje. Ob akcijah na nacionalnem, evropskem in svetovnem nivoju in rezultatih znanstvenih raziskav o hranilni sestavi in ugodnem delovanju čebeljih pridelkov na zdravje, le-ti dodatno pridobivajo na pomenu v vsakdanji prehrani. 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Apidologie, 41: 1-9. https://doi.org/10.1051/apido/2010033 Acta agriculturae Slovenica, 115/2 - 2020 235 AAS_vsebina_2020_115_2_ZOOT_110520.indd 235 16. 06. 2020 09:47:59 AAS_vsebina_2020_115_2_ZOOT_110520.indd 236 16. 06. 2020 09:47:59 doi:10.14720/aas.2020.115.2.562 Original research article / izvirni znanstveni članek Influence of in ovo and pre-starter zinc and copper supplementation on growth performance and gastrointestinal tract development of broiler chickens Vaibhav Bhagwan AWACHAT 1 2, Arumbackam Vijayarangam ELANGOVAN 1 3, Olajide Mark SOGUN-LE \ Corbon Godfrey DAVID \ lyotirmoy GHOSH \ Shivakumar Nisarani Kollurappa GOWDA \ Subrat Kumar BHANJA 4, Samir MAJUMDAR 4 Influence of in ovo and pre-starter zinc and copper supplementation on growth performance and gastrointestinal tract development of broiler chickens Abstract: This experiment was on 350 uniform sized Cobb broiler hatching eggs (60 g) to assess the response of trace mineral supplementation (Zinc and copper) on growth performance and gastrointestinal tract development in broiler chicken. The fertile eggs were divided into groups with in ovo trace mineral solution containing zinc (80 |ig) and copper (16 |ig) and without in ovo administration. After hatching, the chicks were further divided into four groups: Group I served as control without in ovo and without post-hatch supplemented diet (WolNOVO-WoPHS), birds in Group II were without in ovo and with post-hatch supplemented diet (WolNOVO-WPHS) (100 % higher level of zinc 200 ppm, copper 30 ppm in diet), birds in Group III had in ovo (zinc, 80 |ig; copper,16 |g) and without post-hatch supplemented diet (WINOVO-WoPHS) and birds in Group IV had in ovo and with post-hatch supplemented diet (WINOVO-WPHS). Data collected were subjected to completely randomized design. Hatchability, live weight gain, feed intake and feed conversion ratio at 0-3 wk were not affected (p > 0.05) by in ovo administration of the mineral. Post-hatch supplementation of zinc and copper without in ovo supplementation showed better feed conversion ratio at 3-5 wk of age. It could be recommended that for improved post-hatch performance, broiler chickens diets could be supplemented with inorganic zinc and copper. Key words: poultry; broilers; animal nutrition; feed additives; in ovo; trace minerals; growth; gastrointestinal development; immune response Received Septembre 28, 2017; accepted March 06, 2020. Delo je prispelo 28. septembra 2017, sprejeto 06. marca 2020 Vpliv dodatka cinka in bakra v jajce in v krmo po izvalitvi na rast in razvoj prebavil pri brojlerskih piščancih Izvleček: Poskus je bil izveden na 350 valilnih jajcih pi-tovnih piščancev Cobb enotne velikosti, da bi ocenili odziv na dodatek mikromineralov (cink in baker) na rast in razvoj prebavil pri pitovnih piščancih. Oplojena jajca so bila razdeljena v dve skupini, ena je bila tretirana z raztopino cinka (80 |ig) in bakra (16 |g), druga pa ne. Po izvalitvi so bili piščanci razdeljeni v štiri skupine: skupina I je služila kot kontrola brez poseganja v jajce in brez dodatka krmi po izvalitvi (WoINOVO--WoPHS), skupina II ni dobila cinka in bakra v jajce, ampak samo v krmo po izvalitvi (WoINOVO-WPHS), skupina III je dobila cink in baker v jajce, ne pa v krmo po izvalitvi (WI-NOVO-WoPHS), in skupina IV, ki je dobila dodatek cinka in bakra v jajce in v krmo po izvalitvi (WINOVO-WPHS). Zbrani podatki so bili uporabljeni za randomizirano zasnovo poskusa. Valilnost jajc, prirast, zauživanje in izkoriščanje krme v obdobju od izvalitve do treh tednov starosti niso kazali vpliva dodatka cinka in bakra (p > 0,05). Dodajanje cinka in bakra po izvalitvi brez dodajanja v jajca je bilo povezano z boljšim izkoriščanjem krme med 3. in 5. tednom starosti. Za boljše proizvodne rezultate priporočamo dodajanje anorganskega cinka in bakra v krmo za piščance. Ključne besede: perutnina; pitovni piščanci; prehrana živali; krmni dodatki; in ovo; mikrominerali; rast; razvoj prebavil; imunski odziv 1 Indian Centre of Agricultural Research - National Institute of Animal Nutrition and Physiology, Bangalore, Karnataka, India 2 PhD Scholar, ICAR - Indian Veterinary Research Institute, Izatnagar, Bareilly, India 3 Corresponding author, e-mail: avelango@yahoo.co.in 4 Indian Centre of Agricultural Research - Central Avian Research Institute, Izatnagar, Bareilly, India Acta agriculturae Slovenica, 115/2, 237-245, Ljubljana 2020 AAS_vsebina_2020_115_2_ZOOT_110520.indd 237 16. 06. 2020 09:47:59 V. B. AWACHAT et al. 1 introduction The perinatal period is a most crucial time in the development of a young chick as this is a transitional period in which the chicks undergoes metabolic and physiological shifts from the utilization of egg nutrients to exogenous feed (Ferket, 2012). However, with the current work flow of commercial hatcheries and considering time to transport and delivery of newly hatched chicks to broiler farms, the chicks are inevitably exposed to delayed feeding for 48-72 hrs (Noy et al., 2001; Panda et al., 2008; Uni and Smith, 2017). As a consequence of delayed feeding, chicks undergo starvation and allocate the limited reserves of nutrients to the upkeep of ther- Table 1: Ingredient and nutrient composition (%) of experimental diets Post-hatch supplemented Starter Finisher (0-3 days) (4-21 days) (22-35 days) Ingredients (%) Maize 57.53 58.06 62.31 Soybean meal 36.00 36.00 32.00 Sunflower oil 2.00 2.00 2.25 Limestone 1.00 1.00 1.00 Di-calcium phosphate 1.75 1.75 1.50 Salt (NaCl) 0.35 0.35 0.35 L-Lysine HCl 0.59 0.37 0.20 DL-Methionine 0.33 0.22 0.14 L-Threonine 0.20 0.00 0.00 Vitamin/mineral premix * 0.25 0.25 0.25 Analysed Nutrient composition (%) ME (MJ/kg) ** 12.49 12.44 12.74 Crude protein 22.7 22.1 20.5 Lysine 1.68 1.34 1.11 Methionine 0.63 0.50 0.41 Threonine 0.97 0.77 0.73 Arginine 1.40 1.40 1.28 Calcium 1.04 1.04 0.98 Available phosphorus ** 0.45 0.45 0.40 Zinc (ppm) *** 190.3 90.3 88.1 Copper (ppm) *** 29.3 14.3 13.8 * Trace mineral premix 0.1 %, Vit. Premix 0.1 %, B-Complex 0.02 %, Choline 0.05 %. Trace mineral premix supplied mg/kg diet: Mn = 75; Se = 0.2; Fe = 40; Zn = 70; Cu = 10. The vitamin premix supplied per kg diet: Vit. A = 8250 IU; Vit. D3 = 1200 ICU; Vit. K = 1 mg; Vit. E = 40 IU; Vit. Bj = 2 mg; Vit. B2 = 4 mg; Vit. B12 = 1(3 mcg; niacin = 60 mg; pantothenic acid = 10 mg. ** calculated *** Post hatch additional supplemental zinc @ 100ppm and copper @ 15 ppm mal regulation and metabolism which restricts growth and development (Ricklefs, 1987; Pinchasov and Noy, 1993). Delayed feeding causes poor viability and slow growth (Juul-Madsen et al., 2004), increases the weight loss (Bhanja et al., 2015), makes the hatchlings more susceptible to pathogens (Dibner, 1999), restricts the development of critical tissues (Halevy et al., 2000), influences the development of post-hatch gastrointestinal tract maturation (Geyra et al., 2001), increases mortality rate and consequently retards post-hatch growth of day old chicks (Careghi et al., 2005) as pronounced in present day stock of commercial broilers. The development of the gut occurs throughout incubation (Romanoff, 1960), but the functional abilities of the gut only begins to develop about the time the amniotic fluid is orally consumed by the 18th day old embryo. The weight of the intestine, as a proportion of embryonic weight, increases from approximately 1 % at 17 days of incubation to 3.5 % at hatch. Rapid intestinal growth is due to great increase in cell numbers and size, due to accelerated enterocyte proliferation and differentiation and intestinal crypts formation (Uni et al., 2000, Geyra et al., 2001). Therefore, intestinal tissue growth, maturation and metabolism are of great importance in the last period on poultry embryonic development and the early post-hatch period. The sooner the intestine achieves functional capacity, quicker the chicks can utilize dietary nutrients, absorb minerals and vitamins and support the development of skeleton, immune system, breast muscle. The current focus of broiler management needs to be shifted to the fortification of perinatal (last few days of pre-hatch to first few days post-hatch) nutrition so that the early given growth impetus results in achieving the targeted growth in less time. Accordingly, in ovo administration of nutrients in amnion prepares the opportunity for chicks to orally consume supplemented nutrients and develop their digestive and absorptive ability prior to hatch. Growth and development of the embryo and hatchling are dependent on the nutrients in the fertile egg (Richards, 1997). Residual yolk is the main source of nutrients during the transitional period between the hatch and grow-out phases (Gonzales et al., 2003; Henderson et al., 2008). 238 Acta agriculturae Slovenica, 115/2 - 2020 AAS_vsebina_2020_115_2_ZOOT_110520.indd 238 16. 06. 2020 09:48:00 Influence of in ovo and pre-starter zinc and copper supplementation on growth performance and gastrointestinal ... of broiler chickens Micro-minerals that are important to bone formation and strength include Cu, Zn, and Mn, which are greatly reduced in concentration in the egg by the 17th day of incubation (doi) (Yair and Uni, 2011). These minerals also participate through their contribution to enzyme activity along metabolic pathways that are related to the formation of the skeletal system (Bao et al., 2007). Zinc participates in important regulatory pathways for bone and cartilage formation, such as collagen synthesis (Starcher et al., 1980), and hydroxyapatite crystallization (Sauer et al., 1997). Copper is part of the linkage between elastin and collagen, which gives the bone its tensile strength (Carlton and Henderson, 1964). Although Zn is important for collagen synthesis, Cu concentrations must be concomitantly sufficient so that fibrils are not weakened and become susceptible to breakage (Rath, 2000) Currently, copper is added as copper sulphate to pre-mixes of blends for broiler chicken due to its antibacterial properties and to promote the effect of growth. Copper (Cu) is an essential micro element in poultry diets and is required to maintain the proper activities of metalloenzymes associated with iron metabolism. Tyrosinase, oxidase and feroxidase contain Cu, and their activities are dependent on this element, which is an integral part of the cytochrome oxidase system (Wang et al., 2013). Effects of dietary copper-loaded chitosan nanoparticle (CNP-Cu) supplementation on growth performance, haematological and immunological characteristics and the caecal microbiota in broilers were investigated. Results indicated that supplemental CNP-Cu could improve growth performance; affect the immune system (Wang et al., 2013). Varying hatchability with in ovo administration has been reported. There are reports of decreased (Ohta et al., 1999; McGruder et al., 2011) and increased (Bottje et al., 2010) and no effect (Zhai et al., 2011) of hatchability in literature. Therefore, the technique of in ovo administration, nutrient source or dose of nutrients should be perfected to the extent of reducing such loss. Hence, the present study was designed to evaluate if pre-conditioning remains as effective as in ovo supplementation of nutrients in terms of improved growth performance and feed conversion ratio. 2.2 INCUBATION AND IN OVO TREATMENT Three hundred and fifty uniform sized Cobb broiler eggs of 60 g average weight (55-65 g) were procured from commercial hatchery. In the meantime, three hundred and forty-four eggs (98.29 %) were sorted and incubated with the dry bulb temperature ranging from 37.22-37.78 °C and wet bulb temperature of 29.4430.56 °C from days 1 to 18. On day 14, all the unfertile eggs (40 eggs: 88.40 % fertility) were removed after candling. On embryonic day 18, fertile eggs were divided into two groups (152 eggs per group): one without supplementation and another supplemented with in ovo enriched solution containing zinc (80 pg) and copper (16 pg) into the amnion of the embryo under a laminar flow system and then transferred into the hatching trays. The relative humidity was increased by setting the wet bulb thermometer reading of more than 32.22 °C from day 18 till hatching. At hatching, 96.9 % hatchability was from group I (147 hatches) and 87.3 % hatchability was recorded from group II to give 132 hatches. One hundred and thirty-two (132) chicks were then selected per group and further divided into four (66 chicks each) groups; Group I served as control without in ovo and without post-hatch supplemented diet (WoINOVO-WoPHS), Group II composed of hatching eggs without in ovo and with post-hatch supplemented diet (100 % higher level of zinc 200 ppm, copper 30 ppm), Group III composed of hatching eggs with in ovo and without post-hatch supplemented diet. (WINOVO-WoPHS) and Group IV consisted of hatching eggs with in ovo and with post-hatch supplemented diet (WINOVO-WPHS). The required amount of trace minerals were weighed and dissolved in the deionized water in such a concentration that 0.5 ml contained the required amount of trace minerals to be injected in one egg. Before injection, the site was suitably sterilized with 70 % ethanol and the injections were done at the broad end of the egg using 25 mm needle and the pinhole site was sealed with sterile paraffin wax immediately, and eggs were transferred to the hatching trays in the incubator. The entire in ovo procedures were completed within 20 minutes after taking out of eggs from the incubator. 2 materials and methods 2.1 EXPERIMENTAL SITE The animal experimental procedure was approved by ethical committee of ICAR-National Institute of Animal Nutrition and Physiology, Bangalore, India. 2.3 BIRDS AND HOUSING The chicks (immediately from hatchery) from the different treatment groups were randomly distributed into battery cages (6 replicates with 11 chicks in each replicate), fitted with heating arrangements, feeders, wa-terers and dropping trays, with 24 hours light and proper air ventilation, and reared under standard manage- Acta agriculturae Slovenica, 115/2 - 2020 239 AAS_vsebina_2020_115_2_ZOOT_110520.indd 239 16. 06. 2020 09:48:00 V. B. AWACHAT et al. Table 2: Hatchability and chick weight 2.5 MEASUREMENTS Groups Treatments Egg wt (g) Chick wt (%) Hatchability (%) I WoINOVO 59.3 41.3 96.9 II WINOVO 59.4 40.1 87.3 SEM 0.224 0.575 Significance 0.759 0.601 WoINOVO: Without INOVO; WINOVO : With INOVO ment conditions. The temperature inside the cage was maintained at 33 °C on day 1 and gradually reduced to 24-25 °C by the end of the third week and maintained. The feed and fresh drinking water were provided ad libitum during the entire experimental period. Body weight changes were recorded every week to ascertain the weekly and overall body weight gain. The experimental diets were given ad libitum and the residue was weighed at weekly interval in order to arrive at feed intake. Based on the data pertaining to the feed intake and body weight gain, the weekly and period wise cumulative feed conversion ratio (FCR) was calculated. 2.6 GASTROINTESTINAL TRACT DEVELOPMENT 2.4 EXPERIMENTAL DIETS Experimental diets were prepared with maize and soybean meal as major ingredients. The dietary treatments consisted of one normal prestarter diet for group I (WoINOVO-WoPHS) and group III (WINOVO-WoPHS) and one with post-hatch supplemented diet for group II (WoINOVO-WPHS) and group IV (WIN-OVO-WPHS). Ingredient and nutrient composition of experimental diets are given in Table 1. Six birds from each treatment were sacrificed by cervical dislocation at weekly interval (0-4 weeks of age) and twelve birds from each treatment at 5 wk of age (all week data not presented). Gut development was measured by recording the weights of gizzard, proven-triculus, liver as well as weight and length of duodenum, jejunum, ileum and caecum. Immune organ weight (% of live weight) and meat yield (% of live weight) were recorded at the end of the trial. Table 3: Growth performance of broiler chicken Live weight gain (g/b) Feed intake (g/b) Feed conversion ratio 0-3 wk 3-5 wk 0-5 wk 0-3 wk 3-5 wk 0-5 wk 0-3 wk 3-5 wk 0-5 wk Effect of in ovo supplementation (In ovo) WoINOVO 829 1072 1901 1102 1765 2866 1.33 1.65 1.51 WINOVO 843 1089 1932 1106 1807 2913 1.31 1.66 1.51 Significance 0.50 0.66 0.52 0.88 0.35 0.44 0.35 0.66 0.97 Effect of post-hatch supplemented diet (PHS) WoPHS 841 1077 1918 1108 1805 2912 1.32 1.68 1.52 WPHS 831 1084 1916 1100 1767 2867 1.33 1.64 1.50 Significance 0.65 0.85 0.96 0.80 0.40 0.45 0.68 0.28 0.20 Interaction effect (In ovo x phs; WolNOVO-WoPHS 833 1098 1932 1095 1845a 2940 1.31 1.68a 1.52 WoINOVO-WPHS 825 1046 1870 1109 1684c 2793 1.35 1.62b 1.49 WINOVO-WoPHS 849 1055 1904 1120 1765b 2885 1.32 1.67a 1.52 WINOVO-WPHS 838 1123 1961 1092 1849a 2941 1.30 1.66a 1.50 SEM 9.93 18.95 23.43 13.22 24.95 30.46 0.01 0.02 0.01 Significance 0.98 0.13 0.23 0.47 0.01 0.10 0.21 0.01 0.61 a, b, c = Means in the same column bearing different superscripts differ significantly (p < 0.05); WolNOVO-WoPHS = without in ovo and without post-hatch supplemented diet; WoINOVO-WPHS = without in ovo and with post-hatch supplemented diet; WINOVO-WoPHS = with in ovo and without post-hatch supplemented diet; WINOVO-WPHS = with in ovo and with post-hatch supplemented diet; g/b = grams / bird 240 Acta agriculturae Slovenica, 115/2 - 2020 AAS_vsebina_2020_115_2_ZOOT_110520.indd 240 16. 06. 2020 09:48:00 Influence of in ovo and pre-starter zinc and copper supplementation on growth performance and gastrointestinal ... of broiler chickens Table 4: Digestive organ weight (% of live weight) and length (cm/100g live weight) at day 0 Duodenum Jejunum Ileum Caecum Liver Proven-triculus Gizzard Yolk Treatment Length Weight Length Weight Length Weight Length Weight Weight Weight Weight Weight WoINOVO 19.50 1.69 42.37b 2.53b 34.43b 1.84b 8.68b 0.86b 3.03 1.09 9.24 10.05 WINOVO 19.95 1.98 46.21a 2.60a 35.47a 1.88a 10.70a 1.11a 3.24 1.26 9.59 7.13 SEM 0.59 0.08 2.05 0.16 1.49 0.09 0.62 0.07 0.08 0.12 0.35 0.95 Significance 0.18 0.17 0.04 0.04 0.04 0.01 0.01 0.01 0.65 0.37 0.13 0.09 b Means in the same column bearing different superscripts differ significantly (p < 0.05); WoINOVO = Without INOVO; WINOVO = With INOVO 2.7 STATISTICAL ANALYSIS The data were subjected to one way analysis of variance (ANOVA) for completely randomized design and tested for significance between the dietary treatments means employing Tukey's HSD Post-hoc test (SAS, 2010). 3 results 3.1 HATCHABILITY AND CHICK WEIGHT In Table 2, in ovo supplementation of trace mineral enriched solution did not show any significant difference (p > 0.05) in hatchability of in ovo injected group (87.3 %) compared to without in ovo supplementation group (96.9 %). 3.2 GROWTH PERFORMANCE Live weight gain, feed intake and feed conversion ratio during 0-3 wk and overall phase was not affected (p > 0.05) either due to in ovo supplementation of enriched trace mineral solution, post-hatch supplemented diet or their interaction except in 0-3 wk as shown in Table 3. In case of feed intake, there was statistically significant differences only between groups WINOVO-WPHS, WINOVO-W0PHS and Table 5: Digestive organ weight (% of live weight) and length (cm / 100g live weight) at 3rd wk Proven- Duodenum Jejunum Ileum Caecum Liver triculus Gizzard Length Weight Length Weight Length Weight Length Weight Weight Weight Weight Effect of in ovo supplementation (In ovo) WoINOVO 2.84 2.09 6.69 3.53 6.54 3.21 1.38 1.47 2.90 1.25 4.95 WINOVO 2.47 2.40 7.15 3.91 7.06 3.57 1.47 1.74 3.41 1.49 5.32 Significance 0.07 0.18 0.29 0.12 0.17 0.07 0.46 0.18 0.03 0.12 0.39 Effect of post-hatch supplemented diet WoPHS 2.54 2.26 6.85 3.98 6.43 3.66 1.33 1.53 3.27 1.43 5.13 WPHS 2.77 2.22 6.98 3.46 7.17 3.12 1.52 1.68 3.04 1.32 5.13 Significance 0.25 0.75 0.72 0.04 0.15 0.01 0.13 0.68 0.30 0.39 0.90 Interaction effect (In ovo*PHS) WoINOVO-WoPHS 2.69 2.14 6.21 3.57 5.91 3.17 1.37 1.29 2.94 1.29 4.99 WoINOVO-WPHS 2.99 2.03 7.17 3.50 7.17 3.25 1.40 1.64 2.85 1.21 4.90 WINOVO-WoPHS 2.39 2.38 7.50 4.39 6.95 4.14 1.28 1.77 3.60 1.56 5.27 WINOVO-WPHS 2.56 2.42 6.79 3.42 7.17 3.01 1.65 1.72 3.22 1.43 5.36 SEM 0.09 0.12 0.21 0.14 0.22 0.13 0.06 0.11 0.12 0.17 0.09 Significance 0.75 0.63 0.05 0.13 0.33 0.00 0.17 0.55 0.61 0.88 0.90 WoINOVO-WoPHS = Without in ovo and without post-hatch supplemented diet; WoINOVO-WPHS = plemented diet; WINOVO-WoPHS = With in ovo and without post-hatch supplemented diet; WINOVO-supplemented diet : without in ovo and with post-hatch sup-WPHS = With in ovo and with post-hatch Acta agriculturae Slovenica, 115/2 - 2020 241 AAS_vsebina_2020_115_2_ZOOT_110520.indd 241 16. 06. 2020 09:48:00 V. B. AWACHAT et al. WOINOVO-WPHS but not between groups WINOVO-WPHS and WOINOVO-WOPHS. Post-hatch supplemented group without in ovo supplementation showed better feed conversion ratio at 3-5 wk of age. 3.3 DIGESTIVE ORGAN DEVELOPMENT In ovo supplementation significantly (p < 0.05) increased the weight (% of live weight) of jejunum, Ileum and caecum on the day of hatch in in ovo supplemented group compared to un-injected group (Table 4). At 3rd week of age, in ovo supplementation, post-hatch supplemented diet or their interaction groups did not differ significantly (p > 0.05) in all digestive organs weight (% of live weight) and length (cm/100g live weight) except weight of liver, jejunum and ileum (Table 5). Digestive organs length and weight did not show any significant difference at 5th week of age in in ovo supplemented, post-hatch supplemented or their interaction group except weight of duodenum (Table 6). 4 discussion Bakyaraj et al. (2012) reported that hatchability of 81.3 % on in ovo feeding of enriched solution containing zinc 80 pg, copper 16 pg, selenium 0.3 pg and manganese 120 mg/egg compared to sham control group (97.3 %). Dzugan et al. (2014) evaluated effects of the injection of Zn and Cd, individually and in combination and reported that in ovo injection of individual minerals negatively affected hatchability, but had no effect when injected together. Ol-iveira et al. (2015) studied the in ovo injection of commercial diluent containing supplemental micro-minerals (Zn, Mn and Cu) on hatchability and concluded that in ovo injection of higher mineral concentrations into the amnion interfered with embryogenesis during late incubation, due to the creation of a mineral imbalance in the residual amnion. In ovo supplementation of trace mineral enriched solution did not show any significance (p < 0.05) on hatch weight. Oliveira et al. (2015) observed that injection of 0.5 mg of zinc along with manganese and copper did not influence the hatch weight of chicks compared to control. Joshua et al. (2016) also reported that in ovo nano zinc injection at a graded dose (8-20 mg) had no influence on hatch weight. Favero et al. (2013) resulted in no effect on hatchability, hatchling weight and Mn and Cu content in the egg. However, the Zn content in the egg was increased by the substitution. Many of the earlier works (Tako et al., 2005; Goel et al., 2013; Yair et al., 2013; Oliveira et al., 2015) on in ovo injection of trace minerals individually or in combination have not reported increased growth performance of post-hatch Table 6: Digestive organ weight (% of live weight) and length (cm/100 g live weight) at 5th wk Duodenum Jejunum Ileum Caecum Liver Proven- triculus Gizzard Length Weight Length Weight Length Weight Length Weight Weight Weight Weight Effect of in ovo supplementation (In ovo) WoINOVO 1.52 0.99 3.38 1.92 3.22 1.79 0.94 0.82 1.72 0.41 1.93 WINOVO 1.59 1.04 3.31 2.07 3.35 1.72 0.92 0.78 1.74 0.40 2.01 Significance 0.17 0.31 0.57 0.20 0.51 0.55 0.66 0.73 0.58 0.43 0.48 Effect of post-hatch supplemented diet WoPHS 1.54 0.97 3.29 1.97 3.31 1.70 0.92 0.74 1.72 0.39 1.96 WPHS 1.57 1.06 3.39 2.02 3.25 1.80 0.94 0.85 1.74 0.42 1.97 Significance 0.67 0.03 0.54 0.59 0.81 0.23 0.54 0.01 0.78 0.12 0.65 Interaction effect (In ovo x phs; WoINOVO-WoPHS 1.52 0.91 3.40 1.95 3.38 1.81 0.94 0.75 1.72 0.39 2.02 WoINOVO-WPHS 1.51 1.08 3.35 1.89 3.06 1.76 0.94 0.88 1.73 0.44 1.83 WINOVO-WoPHS 1.55 1.03 3.18 2.00 3.25 1.60 0.91 0.73 1.73 0.39 1.90 WINOVO-WPHS 1.63 1.05 3.43 2.15 3.45 1.84 0.93 0.83 1.76 0.41 2.11 SEM 0.03 0.02 0.11 0.05 0.09 0.05 0.02 0.02 0.03 0.01 0.05 Significance 0.93 0.04 0.46 0.34 0.40 0.33 0.86 0.36 0.91 0.59 0.22 WoINOVO-WoPHS = Without in ovo and without post-hatch supplemented diet; WoINOVO-WPHS = plemented diet; WINOVO-WoPHS = With in ovo and without post-hatch supplemented diet; WINOVO-supplemented diet 242 Acta agriculturae Slovenica, 115/2 - 2020 without in ovo and with post-hatch sup-WPHS = With in ovo and with post-hatch AAS_vsebina_2020_115_2_ZOOT_110520.indd 242 16. 06. 2020 09:48:00 Influence of in ovo and pre-starter zinc and copper supplementation on growth performance and gastrointestinal ... of broiler chickens chicks. Joshua et al. (2016) observed a variable result on in ovo injection of graded level of nano zinc, group injected with 40 mg nano zinc showed significant increase in body weight compared to other groups at 5th week. Bakyaraj et al. (2012) reported that in ovo trace mineral supplemented group (Zinc 80 ^g, selenium 0.3 ^g iron 160 ^g, iodine 0.7 ^g per egg) showed significantly higher body weight (411.9) compared to sham control (367.8). In ovo inoculation of several nutrients (maltose, a multi-vitamin supplement, zinc-glycine, glutamine and a mixture containing all these elements and L-carnitine) to 18-day-old embryos did not influence feed intake and feed conversion ratio (dos Santos et al., 2010; Keralapurath et al., 2010; Dooley et al., 2011) The discrepancies in various studies (Ohta and Kidd, 2001; Bhanja and Mandal, 2005; Shafey et al., 2012; Kop-Bozbay et al., 2013; Schulte-Druggelte, 2015) could be explained by many intrinsic and extrinsic factors which affect performance of broiler birds on supplementation of in ovo nutrients. Intrinsic factor of in ovo supplementation includes the content of in ovo solution, pH of solution, os-molarity of solution, dose per egg, site of injection, day of injection, needle bore diameter, interaction effect in mixed two or more nutrients and extrinsic factor include source of hatching eggs, storage condition, weight and size of eggs, nutritive profile of hatching eggs, strain/ line/ breed of breeding birds, breeding age, feeding regimen followed by laying birds, time of hatch. Lack of significant effects in growth performance on in ovo supplementation of trace minerals in this study may be due to ideal level of nutrient present in egg obtained from commercial hatchery. This explanation is supported by the findings of Kop-Bozbay and Ocak (2015) where they found no significant effect of in ovo supplementation of amino acids using eggs with ideal nutrients contents. Most of the researchers did not mention source of hatching eggs especially strain / breed of layer birds used. As in present trial, source of eggs is from commercial hatchery, so neutral effect on growth performance may be related to fast growing broiler strains. This explanation is in line with the findings (Sarica et al., 2009; Yamak et al., 2014; Baeza et al., 2015) that fast-growing birds were better able to perform with commercial basal diet due to the fact that nutrient requirements increase depending on growth rate and also they may be better able to digest the basal diet due to the development of the digestive tract and organs. Furthermore, Schulte-Druggelte (2015) reported that well-nourished, healthy chicks do not respond to in ovo supplements and the degree of limiting protein synthesis of amino acids depend on the ratios and antagonistic relationship between each of these amino acids (Burnham et al., 1992; Dozier III et al., 2011) and the protein content and quality of poultry diets (Ospina-Rojas et al., 2014). The significant difference in digestive organs weights at the 1st week of age supported by the findings of Uni et al. (2003) that in ovo feeding results in improved gastrointestinal tract development of hatchlings and functionally similar to that of conventional 2 day old chicks offered feed immediately after hatch. The authors also indicated that, during the last 3 days of incubation, the weight of the intestine as a proportion of embryo weight increased from approximately 1 % at 17 days of embryonic age to 3.5 % at hatch. In chicks, at 3-7 days of age, the digestive organs will grow at a faster rate as compared to other organs and the small intestine increases in weight more quickly than the body mass during the first week post-hatch (Sklan, 2001). Rapid intestinal growth is due to increase in cell number and size, accelerated enterocyte proliferation and differentiation and intestinal crypt formation (Uni et al., 2000; Geyra et al., 2001). Tako et al. (2005) observed that in ovo injection of Zn-methionine in amniotic fluid on 18th day of incubation increased the vil-lus surface area and enhanced the expression of genes and biochemical activity of intestinal transporters and enzymes thus accelerated intestinal development. Kop-Bozbay and Ocak (2015) observed in experiment of in ovo injection of branched chain amino acids on eggs having ideal levels of nutrient and found that in ovo injection had no effect on the hatchability, chick quality and the degree of growth promotion. Healthy chicks may not respond to in ovo supplements (Schulte-Druggelte, 2015) and the degree of limiting protein synthesis of these amino acids depend on the ratios and antagonistic relationship between each of these amino acids in poultry diet (Burnham et al., 1992) and the protein content and quality of poultry diets (Corzo et al., 2010). The influence of in ovo supplement is greatly dependent on the maternal diet as any deficiency is overcome by extra supplementation. 4 conclusions In ovo supplementation of zinc and copper did not influence hatchability. Birds on the supplementation of zinc and copper recorded better feed conversion ratio at 3-5 weeks of age. In ovo supplementation of zinc and copper significantly increased the weight (% of live weight) of the jejunum, ileum and caecum on the day of hatch. 5 references Baeza, E., Gondret, F., Chartrin, P., Le Bihan-Duval, E., Berri, C., Gabriel, I., . . . Duclos, M. J. (2015). The ability of genetically lean or fat slow-growing chickens to synthesize and store lipids is not altered by the dietary energy source. Animal, 9(10), 1643-1652. https://doi.org/10.1017/S1751731115000683 Bakyaraj, S., Bhanja, S. K., Majumdar, S., & Dash, B. (2012). Acta agriculturae Slovenica, 115/2 - 2020 243 AAS_vsebina_2020_115_2_ZOOT_110520.indd 243 16. 06. 2020 09:48:00 V. B. 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Poultry Science, 90(12), 2681-2688. https://doi.org/10.3382/ps.2011-01532 Acta agriculturae Slovenica, 115/2 - 2020 245 AAS_vsebina_2020_115_2_ZOOT_110520.indd 245 16. 06. 2020 09:48:00 AAS_vsebina_202Q_115_2_ZOOT_110520.indd 246 16. 06. 2020 09:48:00 doi:10.14720/aas.2020.115.2.1345 Original research article / izvirni znanstveni članek Genotype and within-pod bean position microenvironment effect on seed choice for raising cocoa (Theobroma cacao L.) seedlings Babasola Daniel ADEWALE1 2 and Beatrice Abanum NDUKA3 Received November 19, 2019; accepted February 28, 2020. Delo je prispelo 19. novembra 2019, sprejeto 28. februarja 2020. Genotype and within-pod bean position microenvironment effect on seed choice for raising cocoa (Theobroma cacao L.) seedlings Abstract: The probable role of within-pod microenvironment on seed sizes, seedling vigour and biomass yield of four cocoa genotypes was investigated for two years. The respective main, sub and sub-sub plots in the split-split plot experimental design were years, genotypes and within-pod bean positions. Data were taken on cocoa bean length, width and thickness after each pod was opened. Four weekly periodic data were obtained for plant height (PH), stem girth (SG) and number of leaves (NOL); root and shoot biomass yield were also recorded. Analysis of variance revealed significant (p < 0.05) bean position, genotypes, years and some interaction effect on the studied traits. Means of the levels of the three factors differed significantly (p < 0.05). Proximal, middle and distal positions were distinct within-pod microenvironments. The pod middle cavity housed the longest, widest and heaviest beans. Trend analysis of the growing sequences of NOL, PH and SG by the four genotypes differed with bean locations. For bean length, GGE biplot respectively identified CRIN Tc1, CRIN Tc2 and CRIN Tc3 as the best genotype for middle, proximal and the distal positions. The intra-locular space within the pod enhanced differential seed development and maturation; this was evident in the seedling vigour. Key words: bean position; cocoa; micro-environments; GGE biplot; seedling vigour Genotip in mikrookolje glede na položaj semena znotraj ploda vplivata na izbor semen kakavovca (Theobroma cacao L.) za vzgojo sadik Izvleček: Vplivi mikrookolja znotraj plodne glavice na velikost semen, vigor sadik in pridelek biomase so bili preučevani pri štirih genotipih kakavovca v dveh zaporednih rastnih sezonah. Poskus je bil zasnovan na glavnih ploskvah in treh vrstah podploskev kot poskus z deljenkami glede na leto poskusa, genotipe in položaj semen znotraj glavice. Izmerjeni so bili dolžina, širina in višina semen kakavovca potem, ko so se plodovi odprli. Na štiri tedne so bili izmerjeni višina rastlin (PH), obseg debla (SG) in število listov (NOL); izmerjeni sta bili tudi bioma-sa korenin in poganjkov. Analiza variance je pokazala značilno povezavo (p < 0,05) med položajem semen v plodu, genotipom, letom poskusa in nekatere interakcije med preučevanimi znaki. Poprečki vrednosti znakov semen glede na položaj v plodu so se razlikovali statistično značilno (p < 0,05). Proksimalna, srednja in distalna pozicija v plodu so imele značilna mikrookolja. osrednja votlina ploda je imela najdaljša, najširša in najtežja semena. Analiza trendov je pokazala naraščajoče vrednosti znakov NoL, PH in sG za vse štiri genotipe in položaj v plodu. Za dolžino semen so bili na osnovi GGE biplota identificirani genotipi CRIN Tc1, CRIN Tc2 in CRIN Tc3 kot najboljši za srednji, proksimalni in distalni položaj v plodu. Intralokularni prostor v plodu je vzpodbudil diferencialni razvoj semen in njihovo zorenje, kar je bilo očitno tudi na vigorju sejank. Ključne besede: položaj semen; kakavovec; mikrookolje; GGE biplot; vigor sejank 1 Federal University Oye-Ekiti, Ikole-Ekiti Campus, Crop Science and Horticulture, Nigeria 2 Corresponding author, e-mail: d.adewale@gmail.com 3 Agronomy Section, Cocoa Research Institute of Nigeria, Idi-Ayunre, Ibadan, Nigeria Acta agriculturae Slovenica, 115/2, 247-259, Ljubljana 2020 115-2 vsebina.indd 247 23. 06. 2020 07:25:16 B. D. ADEWALE and B. A. NDUKA 1 introduction Cocoa (Theobroma cacao L.), a native crop of South America is well adapted to and flourishes productively in the rainforest ecology of West and Central Africa. The region accounts for the largest proportion of global production, especially from Cote d'lvoire, Ghana, Cameroon and Nigeria. The economic product (i.e. the beans) whose number ranges between 20 to 60 per pod (Ortiz, 2016) are basic raw material for the production of chocolate (Motamayor et al., 2008; Amma et al., 2011). Among the four major cocoa producing member states in the West and Central Africa, production in Nigeria is at the last place. This has grossly being attributed to low yield from most farmers' field. Mathew et al. (2012) identified the use of low quality seed for raising seedlings, low emergence and poor seedling vigour as some of the factors responsible for low productivity within the plantation. In plantation cropping, seedlings obtained from the nursery influences establishment in the field and hence the productivity in the orchard (Bai-yeri, 2006). Therefore, cocoa beans meant for propagation to raise seedlings are expected to have completed their structural and functional development within the pod (the fruit) before they are plucked for use in raising seedlings (Opoku-Ameyaw et al., 2010). The customary practice for cocoa plantation establishment in West and Central Africa has been based on use of seeds in order to generate planting material (Adewale et al., 2016). The observed norm among the farmers has been indiscriminate use of all seeds within the pod irrespective of bean size differences and the location within the pod where they are housed. Seed sizes of genotypes is not only a result of the genetic structure but also some other contributory factors. Seed growth and development is dependent on the biomass investment from the mother plant, such that seeds with high resources have bigger size. Moreover, the endosperm content determines seed sizes which differs significantly within the same fruit (Susko and Lovett-Doust, 2000; Khan et al., 2014). Why should there be significant variation in the seed sizes from the same developmental locations? Khan et al. (2014) identified: within-pod resource quantity, fertilization gradients and neighbor effect to be among some of the probable factors responsible for within-pod seed size variation. Nakamura (1988) identified proximity of the ovules to the stylar end as another important factor in Phaseolus vulgaris L.; noting that seeds closer to the style (proximal) end were significantly better in size than those nearer to the receptacle (the distal) end. Giles (1990) strongly remarked that the within-plant variance cannot be interpreted as anything other than a random environmental variance effect; it is equal- ly obvious that the variation in the sizes of seeds from the same pod is predominantly due to within-pod microenvironment. The fruit (pod) is a controlled environment but within it, there is environmental deviation (Singh and Pokhriyah, 2001) or intra-locular variation due to differential variation in space along the length from one end to the other. The unequal intra-ovary volume/space along the pod length affects and determines the pheno-typic development of the seeds they host in various locations along the length. The fruits provide the environmental space for the seeds and protects the seeds from unfavorable biotic and abiotic condition. However, Bennett et al. (2011) further hinted that the function of the pod to the seeds is far beyond safeguarding them to maturity as an environment, it equally regulates seed growth and maturation. The location/apartment where different seeds appears within the fruit has a role to play in the resource distribution and sharing scheme within the pods (Lee, 1988). In cocoa, Ibikunle (1967) noted that seeds which developed in the expanded (i.e. middle) part of the cocoa pod produced bigger-sized beans and hence seedlings with better vigour. Poor seedling survival at nursery and reduced population of established seedlings on the field are among the attending problem of using all healthy beans from every portions within the cocoa pod. The knowledge of cocoa seedling performance based on positional location within the pod is a needed quest, hence the present investigation, so that beans with no promising vigour will be excluded from use in the propagation scheme. Reports from seed quality test for field establishment of maize (Cruz- Garcia et al., 1995; Moreno-Martinez et al., 1998), barley (Copeland and McDonald, 2001) and rapeseed (Ghassemi-Golezani et al., 2010) noted that low quality seeds produces low vigour seedlings with very poor field establishment. The present study was therefore proposed because there is rare information on the typical role of bean position in the pod on the structural development of the beans. Attempts by Iremiren et al. (2007) and Hammed et al. (2013) were on a single genotype each, hence, genotype and within-pod environment interaction on seedling vigour could not be highlighted in their investigations. Consideration of the housing environment of the cocoa bean as a link to the expression of seedling vigour has not been well attempted in cocoa, hence the need for the present investigation. Highlighting the significance of the bean position within the pod, its relevance to determining bean size and hence, the seedling vigour is worthwhile for identification of portion(s) within the fruit where the most suitable beans capable of supporting good seedling vigour for optimum field establishment is/ 248 Acta agriculturae Slovenica, 115/2 - 2020 115-2 vsebina.indd 248 23. 06. 2020 07:25:16 Genotype and within-pod bean position microenvironment effect on seed choice for raising cocoa (Theobroma cacao L.) seedlings are located. Therefore, the two years replicated experiment which employed four different cocoa genotypes has the following objectives: to identify the role of bean positions within the pod and its interaction with genotypes in the determination of growth and developmental traits of cocoa seedlings. 2 materials and methods With an interest to understand the relative differential performances of cocoa seeds, usually called beans in the different localized positions within the fruit capsules called pods of different cocoa genotypes and the possible link of the same to seedling vigour and development; a research was conducted for two consecutive years at the Cocoa Research Institute of Nigeria (CRIN), Idi-Ayunre, Ibadan, Nigeria. Pods for the experiment were obtained from the hybrid trial plot established in 1999 at the institute. Four physiologically matured cocoa pods were harvested per genotype during the main season (October-November) in three replications in 2014 and 2015. The four hybrids genotypes (CRIN 2011) used for the study were: CRIN Tc1 (T65/7 x N38), CRIN Tc2 (T101/15 x N38), CRIN Tc3 (P7 x PA150), and CRIN Tc4 (T56/7 x T57/22). Each fruit was longitudinally opened and beans were partitioned based on their nearness to the two ends of the pod and the middle as: the proximal (toward the stylar tip), middle (the expanded portion of the fruit) and distal (part closest to the receptacle). An image of cocoa pod delineating the three sections within the pod is shown in Plate 1. Beans within each class were cleaned with sawdust to remove the mucilaginous pulp. Twenty beans were sampled for each of the three positions per genotype and metric measurements on length, width and thickness were taken on the cleaned beans using the venier cali-per following Omokhafe and Alika (2004) and Kaushik et al. (2007). Individual mass of the sampled beans for the three positions of each genotypes were also measured and recorded. The three classified groups of beans per genotype were pre-germinated for 72 hours before they were sown into the polythene bags in the nursery. This was repeated for two years. The experimental design employed was split-split plot design with years, genotypes and bean positions as main, sub and sub-sub plot factors respectively. The number of replications used was three. Among the measurements taken on the seedlings after germination were: number of leaves per plant, plant height and stem girth. Data on the morphological characteristics continued from 2nd weeks after seedling emergence to the sixth month. Destructive sampling was done for the sampling unit after the termination of the experiment to obtain the fresh and dry root, shoot and total biomass yield. The data were subjected to analysis of variance (ANOVA) and means of the different main effects were separated by Tukey's honestly significant differences. The association between the bean indices and the harvested biomass after destructive sampling were tested by correlation analysis. All analysis were carried out in SAS (version 9.4, 2011). Furthermore, trend analysis was done using R Development Core Team (2013) to understand the sequence of response of the growth data taken at intervals. Traits with significant genotype by bean position interactions from the ANOVA were further partitioned using the "which won where" option in the GGE biplot in GEA-R (Pacheco et al., 2016). From the component of the ANOVA, genetic estimates were calculated for phenotypic and genotypic coefficients of variation (PCV Plate 1: The three cross-sections of an opened cocoa pod housing cocoa beans Acta agriculturae Slovenica, 115/2 - 2020 249 115-2 vsebina.indd 249 23. 06. 2020 07:25:16 B. D. ADEWALE and B. A. NDUKA and GCV) following the method of Singh and Chaudhay (1999): PCV = (a2p / X)*.......................................Eq. 1 GCV = (a2g / X)*...............................................Eq. 2 Where a2p> a2g and X are phenotypic and genotypic variances and grand mean respectively. Broad sense heritability (Hbs) was expressed as the percentage of the genotypic variance to the phenotypic variance for split-split plot design as described by Bok-meyer et al. (2009), cited in Clark and Watkins (2012) and modified as follows: Hbs = a2 / a2 + a2 . + a2 . + a: + a2 a2 gy/y gp/p gr(y)/rp gyp/yp + e/ryp ..Eq. 3 Repeatability (rc) was estimated following Ortiz and Ng (2000), as follows: r a2 /(a2 + a2 ) c = g v y gy' 3 results ..Eq. 4 Table 1 shows the significant (p < 0.05) differences in the bean metric traits for years, varieties, within-pod Table 1: Variance components for the bean metric traits, correlations among them and their mean performances based on years, varieties and bean position Sources of Variation DF BL (cm) BW (cm) BT (cm) BM. (g) Replications 2 0.04 0.03 0.02 0.08 Years 1 4.86*** 0.03 1.34*** 5.08*** Error (a) 2 0.05 0.03 0.04 0.05 Varieties 3 3.36*** 2.89*** 2.24*** 8.42*** Years*Varieties 3 0.98*** 0.52*** 0.42*** 3.69*** Error (b) 12 0.04 0.03 0.03* 0.06 Bean Positions 2 1.35*** 0.62*** 0.56*** 1.23*** Years* Bean Positions 2 0.87*** 0.06 0.04 0.58*** Varieties* Bean Positions 6 0.27*** 0.07** 0.36*** 0.45*** Years*Varieties* Bean Positions 6 0.35*** 0.04 0.15*** 0.57*** Error (c) 32 0.05 0.02 0.02 0.08 Correlations among the three bean metric traits with bean mass Bean mass 0.96ns 0.99* 0.99** - Mean separation of the three main effects Years 2014 2.11b 1.14a 0.65b 1.68a 2015 2.31a 1.13a 0.76a 1.48b Varieties CRIN Tc 1 2.37a 1.27a 0.83a 1.89a CRIN Tc 2 2.34a 1.26a 0.81a 1.71b CRIN Tc 3 2.07b 0.95c 0.53c 1.33c CRIN Tc 4 2.06b 1.06b 0.66b 1.39c Bean Positions Proximal 2.13b 1.11b 0.75a 1.53b Middle 2.31a 1.20a 0.64b 1.68a Distal 2.19b 1.08b 0.73a 1.53b t BL - Bean Length, BW - Bean Width, BT - Bean Thickness, BM. - Bean mass. ** and *** - Significance at p = 0.05, 0.01 and 0.001 Mean comparison is along the column for year, varieties and bean position; means with the same alphabet are not significantly different from each 250 Acta agriculturae Slovenica, 115/2 - 2020 115-2 vsebina.indd 250 23. 06. 2020 07:25:16 Genotype and within-pod bean position microenvironment effect on seed choice for raising cocoa (Theobroma cacao L.) seedlings bean positions, years by varieties, years by bean positions and varieties by bean position interactions. Bean length and its thickness differed significantly (p < 0.05) among the two years and higher significant values occurred in 2015. However, bean mass was significantly (p < 0.05) higher (1.68 g) in 2014 compared to 1.48 g in 2015 (Table 1). Beans length, width and thickness were highest for CRIN Tc1 and 2, but minimum values for the same were obtained in CRIN Tc3. The four metric traits (length, width, thickness and mass) of the beans varied significantly (p < 0.05) with positions where the beans were located within the pod. Beans in the middle posi- tion had significant (p < 0.05) longer, wider and heavier beans mass when compared to beans at the proximal and distal positions (Table 1). Furthermore in Table 1, individual bean mass had strong to very strong and significant (p < 0.01) correlation with bean width (r = 0.99) and thickness (r = 0.99). Table 2 shows different sources of variation in the analysis of variance and the mean values of some traits at the termination of the experiment for the beans from the three within-pod environments for the four genotypes in the two years. The fresh shoot mass of the cocoa seedlings was not significantly enhanced in this study (Table Table 2: Variance components for the seedling biomass traits, correlations among them and their mean performances based on years, varieties and bean position Sources of Variation DF SFM SDM RFM RDM Replications 2 4.2 2.47 1.24 1.02*** Years 1 6.73 25.32*** 0.31 1.89*** Error (a) 2 10.03 0.07 14.42 0.04 Varieties 3 31.32* 1.98* 2.85* 0.49*** Years*Varieties 3 7.47 0.23 3.68* 0.01 Error (b) 12 8.27 0.18 1.07 0.04 Bean Positions 2 11.05 1.74 4.91** 0.46*** Years* Bean Positions 2 14.72 0.45 3.68* 0.01 Varieties* Bean Positions 6 6.29 0.44 2.362 0.12 Years*Varieties* Bean Positions 6 11.34 0.69 1.35 0.06 Error (c) 32 6.02 0.64 1.04 0.06 Correlations among the fresh and dried biomass of the shoot and root Shoot Dry Mass 0.99** - 0.88ns 0.95ns Mean separation of the three main effects Years 2014 9.90a 2.51b 4.69a 0.79b 2015 10.51a 3.69a 4.56a 1.12a Varieties CRIN Tc 1 10.87ab 3.56a 5.20a 1.19a CRIN Tc 2 8.73b 2.77b 4.37b 0.93b CRIN Tc 3 9.54ab 2.98ab 4.36b 0.89b CRIN Tc 4 11.67a 3.09ab 4.58b 0.81b Bean Positions Proximal 9.91a 2.99a 4.23b 0.86b Middle 10.98a 3.41a 5.12a 1.11a Distal 9.72a 2.90a 4.53ab 0.89b t SFM - Shoot fresh mass, SDM - Shoot dry mass, RFM - Root fresh mass, RDM - Root dry mass*, ** and *** - Significance at p = 0.05, 0.01 and 0.001 Mean comparison is along the column for year, varieties and bean position; means with the same alphabet are not significantly different from each other Acta agriculturae Slovenica, 115/2 - 2020 251 115-2 vsebina.indd 251 23. 06. 2020 07:25:16 Table 3: Mean performances of the vegetative growth traits across different years varieties and bean positions in the pod NOL NOL NOL NOL NOL PH PH PH PH PH SG SG SG SG SG 6WAS IOWAS 14WAS 18 WAS 22WAS 6WAS IOWAS 14WAS 18 WAS 22WAS 6WAS IOWAS 14WAS 18 WAS 22WAS Year 2014 4.75b 6.89a 8.80a 10.68a 13.22a 16.43a 19.63a 21.82a 25.67a 27.43a 0.40a 0.58a 0.63b 0.67a 0.92a 2015 5.23a 7.27a 8.53a 9.66b 11.86a 14.89b 18.36b 21.82a 23.41b 27.36a 0.22b 0.45b 0.66a 0.74a 0.82a Varieties CRIN Tc 1 5.16ab 7.23ab 8.52b 9.99ab 12.32a 16.25a 20.84a 23.77a 25.92a 28.18a 0.31a 0.45a 0.66a 0.75a 0.89a CRIN Tc 2 4.53c 6.35c 7.81b 9.37b 12.09a 14.65b 17.40b 19.56b 22.95b 26.71b 0.28a 0.57a 0.64a 0.74a 0.81a CRIN Tc 3 4.70bc 6.88bc 8.60b 10.29ab 12.56a 14.52b 16.88b 19.30b 22.58b 25.56b 0.33a 0.61a 0.70a 0.76a 0.90a CRIN Tc 4 5.56a 7.87a 9.72a 11.03a 13.19a 17.21a 20.86a 23.70a 26.69a 29.11a 0.33a 0.53a 0.56a 0.68a 0.89a Cocoa Beans Position Proximal 4.86a 6.80a 8.44a 9.86ab 12.34ab 15.71ab 18.96ab 21.5b 24.22b 27.52ab 0.34a 0.59a 0.65a 0.83a 0.98a Middle 5.13a 7.31a 9.13a 10.89a 13.42a 16.36a 19.78a 22.78a 25.83a 28.65a 0.34a 0.53a 0.59a 0.65a 0.82a Distal 4.97a 7.14a 8.42a 9.75b 11.85b 14.91b 18.24b 20.46b 23.56b 26.00b 0.25a 0.51a 0.71a 0.81a 0.81a NOL-Number of leaves, PH- Plant height, SG-Stem Girth, WAS- weeks after sowing Mean comparison is along the column for years, varieties and bean positions; means with the same alphabet are not significantly different Genotype and within-pod bean position microenvironment effect on seed choice for raising cocoa (Theobroma cacao L.) seedlings Table 4: Some genetic estimates of some variables Traits PCV GCV GCV:PCV Heritability (%) Repeatability Bean length 134.63 123.30 91.59 83.88 0.77 Bean width 167.87 159.92 95.26 90.75 0.85 Bean thickness 192.70 178.89 92.83 86.17 0.84 Bean mass 258.05 230.85 89.46 80.03 0.70 Shoot fresh mass 198.76 175.23 88.16 77.73 0.81 Shoot dry mass 88.21 79.92 90.60 82.08 0.90 Root fresh mass 112.68 78.46 69.63 48.48 0.44 Root dry mass 75.88 71.44 94.15 88.64 0.98 NOL6WAS 95.90 87.95 91.71 84.11 0.82 N0L10WAS 107.46 101.40 94.37 89.05 0.92 N0L14WAS 119.43 113.76 95.25 90.73 0.97 N0L18WAS 104.27 91.69 87.93 77.33 0.76 N0L22WAS 96.80 56.48 58.35 34.04 0.29 PH6WAS 153.71 139.67 90.87 82.57 0.73 PH10WAS 216.38 209.40 96.77 93.65 0.91 PH14WAS 241.21 227.25 94.21 88.76 0.83 PH18WAS 189.17 177.50 93.83 88.05 0.82 PH22WAS 166.93 127.03 76.10 57.91 0.50 SG6WAS 53.21 17.96 33.75 11.39 0.11 SG10WAS 94.86 58.25 61.41 37.71 0.41 SG14WAS 126.20 88.47 70.10 49.14 0.47 SG18WAS 56.98 29.07 51.01 26.02 0.23 SG22WAS 56.67 18.57 32.77 10.74 0.10 NOL-Number of leaves, PH- Plant height, SG-Stem girth taken at four weeks interval from the 6th to the 22nd weeks after planting 2), however, the dry shoot mass shown in 2014 had a lead and significant (p < 0.05) value of 3.69 g compare to 2.51 g in 2015; the trend was same for the dry root mass (Table 2). The middle positional beans produced the highest significant (p < 0.05) mean for root fresh and dry mass. Neither year nor the three beans position inside the cocoa pod affected the shoot fresh mass. However, among the varieties, the highest fresh and dry root mass value was observed in CRIN Tc1. Moreover, shoot fresh and dry mass had a strong (0.99) and significant (p < 0.01) correlation (Table 2). From the means in Table 3, significantly (p < 0.05) higher mean was obtained for NOL6 weeks after sowing (WAS) and SG14WAS in 2015. However, higher significant (p < 0.05) values were obtained for NOL-18WAS, PH6WAS, PH10WAS, PH18WAS, SG6WAS and SG10WAS in 2014 (Table 3). Among the four genotypes, CRIN Tc1 and CRIN Tc4 had the significantly (p < 0.05) higher means for number of leaves and plant heights at 6th to the 22nd WAS (Table 3). Beans originating from the middle of the pod produced significantly (p < 0.05) the highest number of leaves (18 and 22 WAS) and plant height (6th to 22nd WAS) in Table 3. For all the variables studied in this experiment, the phenotypic coefficient of variation were higher than the genotypic coefficient of variation (Table 4). The proportion of the genetic component in the phenotypic coefficient of variation ranged between 32.77 (SG22WAS) to 96.77 (PH10WAS). Stem girth 22 WAS which had the lowest GCV: PCV, equally had the lowest broad sense heritability (10.74) and repeatability (0.10). The highest (90.75) broad sense heritability occurred in bean width while the highest (0.98) repeatability was recorded for root dry mass (Table 4). Table 5 unveiled the specific pattern of variability and sequence of response of each of the four genotypes to three vegetative and agronomic variables. The sources of variation from the table includes: the total treatment, each of the two factors within the treatment (i.e. intervals and bean position) and variability based Acta agriculturae Slovenica, 115/2 - 2020 253 115-2 vsebina.indd 253 23. 06. 2020 07:25:17 B. D. ADEWALE and B. A. NDUKA Table 5: Trend analysis of the growth traits measured at intervals in correspondence to the three bean positions within the pod for four cocoa varieties CRIN Tc 1 CRIN Tc 2 Sources of Variation DF NOL PH SG NOL PH SG Mean Squares Mean Squares Treatments 14 7.27*** 18.14*** 0.11 6.99** 17. 49*** 0.06 Interval (In) 4 24.54*** 58.51*** 0.24* 23.84*** 55.55*** 0.11 In-Linear 1 66.86*** 190.71*** 0.35* 67.02*** 176.61*** 0.18 In-Quadratic 1 1.70** 7. 45** 0.15 7.17* 0.55 0.03 In-Cubic 1 1.15* 2.08 0.05 5.7* 0.03 0.01 In-quantic 1 0.05 0.20 0.01 1.89 0.67 0.14 Bean Position(BP) 2 1.17* 8.13** 0.06 0.58 10.35*** 0.03 BP-Linear 1 0. 48 0.29 0.10 0.60 18.66*** 0.05 BP-Quadratic 1 1.87** 15.98*** 0.03 0.57 12.02* 0.01 Error 8 0.15 0. 46 0.05 0.17 0.24 0.04 CRIN Tc 3 CRIN Tc 4 Sources of Variation DF NOL PH SG NOL PH SG Mean squares Mean Squares Treatments 14 9.13** 19.17** 0.04 7.22** 20.05*** 0.03*** Interval(In) 4 28.09*** 62.76*** 0.04 24.26** 63.12*** 0.11*** In-Linear 1 83.66*** 189.68*** 0.03 75.19*** 206. 46*** 0.28*** In-Quadratic 1 0.29 2. 46 0.02 0.03 1.09 0.12*** In-Cubic 1 0.54 0.14 0.04 0.31 0.56 0.004** In-quantic 1 0.001 0.34 0.001 0.04 0.17 0.003* Bean Position(BP) 2 5. 49* 3.15 0.01 1.31** 10.35** 0.0003 BP-Linear 1 2.96* 0.98 0.002 7.38* 3.23 0.00007 BP-Quadratic 1 7.99** 5.32 0.02 7.56* 17. 49** 0.0006 Error 8 0.55 1.39 0.04 0.17 0.93 0.0003 Note. DF - Degree of freedom, *,**,and*** - significance at p- 0.05, 0.01 and 0.001, NOL - Number of leaves, PH -Plant height; SG - Stem girth on different forms of trend for each of the two factors. Highly significant (p < 0.01) variabilities were noted for the fifteen treatment combinations and the five intervals of data measurements for number of leaves, plant height and stem girth of the four genotypes. However, there was significant (p < 0.05) treatments effect on stem girth for CRIN Tc4 while interval effect was equally significant (p < 0.05) for CRIN Tc1 and 4 for stem girth (Table 5). With respect to bean position as a source of variation, its effect was significantly (p < 0.05) notable for number of leaves and plant height for CRIN Tc1 and 4; CRIN Tc2 and 3 showed respective significance (p < 0.05) for plant height and number of leaves (Table 5). Number of leaves showed linear (p < 0.001), quadratic (p < 0.01) and cubic (p < 0.05) response for CRIN Tc1 and CRIN Tc2. Moreover, trend for plant height was linear (p < 0.001) and quadratic (p < 0.001) for CRIN Tc1 but only linear for CRIN Tc2 while the trend response for stem girth was only linear (p < 0.05) for CRIN Tc1 (Table 5). Within the same table, CRIN Tc3 and CRIN Tc4 showed only significant (p < 0.05) linear response for number of leaves and plant height. Specifically, the stem girth displayed significant (p < 0.05) linear to quantic responses for CRIN Tc4 (Table 5). Still from Table 5, the trend response with respect to bean position for plant height was both linear (p < 0.05) and quadratic (p < 0.05) in CRIN Tc2, number of leaves and plant height displayed quadratic (p < 0.05) response in CRIN Tc1 and CRIN Tc4. However, for CRIN Tc3 and CRIN Tc4, the number of leaves exhibited both linear (p < 0.05) and quadratic (p < 0.05) responses. Factors 1 and 2 (Figure 1) cumulatively explained the total variance. The proximal, the middle and the distal position were three distinct within-pod environments (each appearing in different sector) for cocoa bean length 254 Acta agriculturae Slovenica, 115/2 - 2020 115-2 vsebina.indd 254 23. 06. 2020 07:25:17 Genotype and within-pod bean position microenvironment effect on seed choice for raising cocoa (Theobroma cacao L.) seedlings Figure 1: Bean position by varieties interaction display for cocoa bean length 1 - CRIN Tc1, 2 - CRIN Tc2, 3 - CRIN Tc3 and 4 - CRIN Tc4 I \ r i r _- r"'-'----------------.-^c- Pranrfial -------2------- 1-1--1-T" •1.0 -0.5 0.0 0.5 1.0 Factor 1 (99.97%) Figure 2: Bean position by varieties interaction displayed for cocoa bean thickness 1 - CRIN Tc1, 2 - CRIN Tc2, 3 - CRIN Tc3 and 4 - CRIN Tc4 determination. Each of the three distinct within-pod microenvironments identified different best performing genotype; such that CRIN Tc1, CRIN Tc2 and CRIN Tc3 respectively had the best bean length in the middle, proximal and the distal portion within the pod (Figure 1). Only two sectors were prominent in Figure 2 and the three within-pod positions differentially dispersed within the major sector. However, CRIN Tc1, CRIN Tc3 and CRIN Tc4 were respectively the vertex genotype for distal, proximal and middle within-pod microenvironment respectively for cocoa bean thickness determination. Figure 3 displayed the bean position by varieties interaction for cocoa bean width. The three within-pod locations were distinct for bean width determination. The trapezia polygon had four sectors which identified CRIN Tc1 as the vertex genotype for beans at the middle cavity, CRIN Tc3 for the distal and CRIN Tc4 for the proximal within-pod location (Figure 3). The polygonal display of the bean position by varieties interaction for cocoa bean mass was a triangle with three sectors (Figure 4). The proximal and the distal within-pod microenvironments were accommodated in one mega environment while the middle portion of the pod environment was alone in another sector as another mega environment. The vertex genotype for both the proximal and distal within-pod environment was CRIN Tc3, but the sector which captured the middle environment had CRIN Tc4 as the vertex genotype (Figure 4). 3 discussion The performances of the four bean metric measurements and seedling vegetative traits were affected by the Acta agriculturae Slovenica, 115/2 - 2020 255 115-2 vsebina.indd 255 23. 06. 2020 07:25:17 B. D. ADEWALE and B. A. NDUKA year effect. This seem to reveal that the variables are not stable but very plastic as they significantly responded to changes in the wider environment of yearly climatic variation. The four studied genotypes and the three bean positions equally distinguished themselves on the four beans metric and other vegetative traits. This further substantiate that character expression is dependent on the environment, the genotype and the interaction of both (Crossa et al., 1991; Mortazavian and Azizinia, 2014). Moreover, by this study, the two different years (2014 and 2015), the four cocoa varieties (CRIN Tc1, CRIN Tc2, CRIN Tc3 and CRIN Tc4) and the three bean positions (proximal, middle and distal) were unique treatments in the experiments. The environment within the ovary is not consistently uniform, hence, compartments within the seed-developing space do impacts and determines the physical and physiological traits of the seed (Illipronti et al., 2000). The spacious hollow at the middle cavity of the cocoa pod may have supported the recorded bean features of longer length, wider width and heavier mass of the beans which developed in the area. It is clear from our result that the length and width of the bean rather than the thickness were noted to be more important in bean mass determination. We equally noted that beans from the middle position supported increased biomass yield of roots and shoots for the four cocoa genotypes. In Cryptocarya alba (Molina) Looser, large sized seeds were associated with larger shoots, roots and number of leaves (Chacon et al., 1998). The reduced value for the same traits at the proximal and distal ends could be due to some constriction of the hollow within the pods. However, beans at the two ends were significantly thicker than those in the middle of the cavity. This seems to infer that adequate space is very necessary for efficient seed development in the ovary. The tapering structure of the pod at proximal and distal ends may be very key in the reduction on the sizes of the seeds located in and towards the two ends. So, the available space within the pod (which varies along its length) could be a determinant of the seed sizes and hence mass of beans in the cocoa pods. This result is in consonance with the report of Iremiren et al. (2007) and Hammed et al. (2013) on their work with a genotype called F3 Amazon. Following the descriptive pod and pod apex shape by Phillip-Mora et al. (2013), the pod shape of the four genotypes used in this study was more similar to 'Angoleta' with the pod apex ranging between acute to obtuse. However, for whichever shape the cocoa pod has, the middle part (which is usually raised or expanded) seem to provide wider space for bean development compare to the two tapering ends. The identified significant within-pod variation which leads to three group of beans from the same pod observed in this study may not be true for 'Calabacillo' pod shaped cocoa (Phillip-Mora et al., 2013) which are usually ball-like round. Contrary to the report of Perin et al., (2002) on melon (Cucumis melo L.) where stems are the organs mostly affected by seed size, our result negates their assertion, the significant differences in the metric traits on the beans did not affect cocoa seedlings stem girth as it does to other vegetative traits. Genotypes are genetic entity with specific characteristics which makes them different from another one. Bekele et al (2006) had much earlier noted that there exist considerable genetic variation in fruit size, shape and bean size of cocoa. The four cocoa genotypes used in this study differed in sizes of their beans and the impact of each of the bean traits on vegetative and biomass yield Figure 3: Bean position by varieties interaction display for cocoa bean width 1 - CRIN Tc1, 2 - CRIN Tc2, 3 - CRIN Tc3 and 4 - CRIN Tc4 256 Acta agriculturae Slovenica, 115/2 - 2020 115-2 vsebina.indd 256 23. 06. 2020 07:25:17 Genotype and within-pod bean position microenvironment effect on seed choice for raising cocoa (Theobroma cacao L.) seedlings —i-1--1-r~ -1.0 -0.5 0.0 0.5 1 0 Factor 1 (66.03 %> Figure 4: Bean position by varieties interaction display for cocoa bean mass 1 - CRIN Tc1, 2 - CRIN Tc2, 3 - CRIN Tc3 and 4 - CRIN Tc4 of the four cocoa genotypes; this information is a useful resource on which selection programme on wet bean sizes can thrive. Our research outcome conforms to the expected norm that the phenotypic variance component are always higher than the genotypic component. However, variables which shows very small deviation are remarked to be reliable traits (Adewale et al., 2010), because the proportion which accrued to environmental variation is small while the quantity of variation for the genetic portion is high. The observed high genetic components in the phenotypic expression of the four bean metric traits, number of leaves and plant height measured at different interval in this study is remarkable. High and positive correlation existed between broad sense heritability and repeatability, revealing that traits with high broad sense heritability will have high repeatability. It is noteworthy that traits with high repeatability may have low response to environmental variation, hence the corresponding high broadsense heritability could be due to additive gene action. Our research considered two notable environments: the within-pod environment (a micro environment) and the year (a macro environment), both which affected the expression of the bean metric traits and vegetative characteristics differently. GGE biplot identified the proximal, middle and distal positions within the pod environments for cocoa bean length, width, thickness and mass to be very unique. Hammed et al. (2013) who observed this three distinct divisions along the cocoa pod length, like us, did remark that the beans in the middle were longer, wider, thicker and heavier than other beans from the two extreme ends of the pod. For the three metric measurements on the bean, the GGE biplot clearly distinguish bean sizes, noting that the length, width and thickness of beans differ in respect to the positions where they are located along the inner cavity of the pod length. This therefore infers that the environment where beans are located during development primarily determines the phenotypic expression of its length, width, thickness and mass. The significant differences for preference of the four genotypes for the various within-pod location is a reflection of genotypic variation. From this study therefore, mass of individual beans did not differ at proximal and distal positions, hence the zoning of the two microenvironments as a single megaenvironment by GGE biplot for the trait. This further denotes that the mass of beans from the two ends of the cocoa pod do not differ from each other; meaning that the variation between both for mass of individual bean was not significant enough (Yan and Kang, 2003). Furthermore, the relevance of the individual bean mass of the two positions (proximal and distal) within the cocoa pod is the same. The long-standing recommendation of utilizing Cocoa beans from the middle cavity rather than the two ends (Ibikunle, 1967; Iremiren et al., 2007; Hammed et al., 2013) may have stemmed from the conspicuous variation in bean size and mass among beans from the same pod coupled with the linear correspondence between heavier seeds and high seedling vigour (Enayatgholizadeh et al., 2011). 4 conclusion Our work clearly revealed that the three inner locations (proximal, middle and distal) along cocoa pods length are prominent in distinguishing cocoa bean sizes; identifying the cocoa beans in the middle of the pod to be of the highest quality for all the metric measurements Acta agriculturae Slovenica, 115/2 - 2020 257 115-2 vsebina.indd 257 23. 06. 2020 07:25:17 B. D. ADEWALE and B. A. NDUKA including bean mass. We suggest consideration of the influence of pod position on the cacao tree in subsequent related work to assess its probable influence on bean sizes relative to within pod positions. Where adequate bean may not be available for seedling generation, seeds from the two ends of the pod may not be discarded as seedlings from them improved in their growth with active photosynthesis after establishment. However, selection of and usage of beans in the middle of the pod could lead to the production of good, uniform and vigorous seedlings that will support higher cocoa productivity. 5 acknowledgement The authors wish to appreciate the support from the Agricultural Superintendents and the Nursery Attendants at the Nursery section of the Cocoa Research Institute of Nigeria (CRIN), Idi-Ayunre, Ibadan, Nigeria during the research. 6 references Adewale, B.D., Okonji, C., Oyekanmi, A.A., Akintobi, D.A.C. and Aremu, C.O. (2010). Genotypic variability and stability of some grain yield components of cowpea. African Journal of Agricultural Research, 5, 874-880. Adewale, B.D., Adeigbe, O.O. and Muyiwa, A.A. (2016). 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(1990). The effects ofvariation in seed size on growth and reproduction in the wild barley Hordeum vulgare ssp. spontaneum. Heredity, 64, 239-250. https://doi.org/10.1038/ hdy. 1990.29 Hammed, L.A., Olaiya, A.O., Lawal, I.O., Idowu, O.T.H. and Aiyelaagbe, I.O.O. (2013). Effects of some bean characters on germination and seedling growth of cocoa (Theobroma cacao L.). Nigerian Journal of Horticultural Sciences, 17,126134. Ibikunle, B.O. (1967). Effect of position of beans in the pod on the performance of cocoa seedlings. Cocoa Research Institute of Nigeria Annual Report. Illipronti Jr., R.A., Lommen, W.J.M., Langerak, C.J. and Struik, P.C. (2000). Time of pod set and seed position on the plant contribute to variation in quality of seeds within soybean seed lots. Netherlands Journal of Agricultural Sciences, 48, 165-180. https://doi.org/10.1016/S1573-5214(00)80012-3 Iremiren, G.O., Famaye, A.O. and Oloyede, A.A. (2007). 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Pesquisa Agropecuária Brasileira, 37, 1711-1718. https://doi.org/10.1590/S0100-204X2002001200006 Phillips-Mora, W., Arciniegas-Leal, A., Mata-Quiros, A. and Motamayor-Arias, J.C. (2013). Catalogue of cacao clones selected by CATIE for commercial plantings, Turrialba. R Development Core Team. (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. Austria. URL Retrieved from http:// www.R-project.org/. SAS. (2011). SAS Online Doc® 9.4. SAS Institute Inc., Cary, NC. Singh, R.K. and Chaudhary, B.D. (1999). Biometrical methods in quantitative genetic analysis. New Delhi: Kalyani Publisher. Singh, N. and Pokhriyal, T.C. (2001). Variations in pod and seed traits in six different Dalbergia sissoo seed sources. Journal of Tropical Forest Science, 13, 162-170. Susko, D.J. and Lovett-Doust L. (2000). Patterns of seed mass variation and their effects on seedling traits in Alliaria peti-olata (Brassicaceae). American Journal of Botany, 87, 56-66. https://doi.org/10.2307/2656685 Yan, W. and Kang, M.S. (2003). GGE biplot analysis: a graphical tool for breeders, geneticists and agronomists. Boca Raton, Florida Fl: CRC Press. https://doi. org/10.1201/9781420040371 Acta agriculturae Slovenica, 115/2 - 2020 259 115-2 vsebina.indd 259 23. 06. 2020 07:25:17 115-2 vsebina.indd 260 23. 06. 2020 07:25:17 doi:10.14720/aas.2020.115.2.1359 Original research article / izvirni znanstveni članek Intensification of the drying process of small seed oilseeds using microwave electromagnetic radiation Ildar GANEEV 1 2, Khasan KARIMOV \ Shamil FAYZRAKHMANOV \ Ilgam MASALIMOV \ Valeri PERMYAKOV 1 Received November 28, 2019; accepted March 3, 2020. Delo je prispelo 28. novembra 2019, sprejeto 03. marca 2020 Intensification of the drying process of small seed oilseeds using microwave electromagnetic radiation Abstract: One of the important and crucial stages of post-harvest treatment of rapeseed is drying. The purpose of the article is to improve the drying process of seeds of small seed oil crops using electromagnetic radiation of the microwave range in order to increase its productivity and determine the optimal operating parameters. The article describes the construction of a new microwave (UHF) dryer with a capacity of 200 kg h-1 for drying small-seeded crops. Curves were obtained that show the dependence of the heating temperature of seeds on microwave power, the effect of initial seed moisture and heating temperature on drying kinetics. The ratio of the stages of microwave heating and cooling was determined, which allows to increase the drying efficiency. Key words: grain drying; rapeseed; electromagnetic radiation; drying kinetics; drying device Pospeševanje sušenja majhnih semen oljnih poljščin z mikrovalovnim elektromagnetnim sevanjem Izvleček: Eden od pomembnejših in ključnih postopkov pri požetveni obravnavi semen oljne ogrščice je sušenje. Namen prispevka je izboljšanje procesa sušenja majhnih semen oljnih rastlin z mikrovalovnim elektromagnetnim sevanjem z namenom povečanja produktivnosti in določiti optimalne operacijske parametre. Članek opisuje zgradbo novega mikrovalovnega (UHF) sušilnika z zmogljivostjo 200 kg h-1 za sušenje majhnih semen oljnih poljščin. Krivulje kažejo odvisnost temperature segretih semen od moči mikrovalovnega sušilnika, učinka začetne vlažnosti semen in odvisnost sušilne temperature od kinetike sušenja. Določeno je bilo razmerje med gretjem in hlajenjem mikrovalovnega sušilnika, ki omogoča povečanje učinkovitosti sušenja. Ključne besede: sušenje zrnja; oljna ogrščica; elektromagnetno sevanje; kinetika sušenja; sušilnik 1 Federal State Budgetary Educational Establishment of Higher Education "Bashkir State Agrarian University", Department of Mechanics and machine construction, Ufa, Russia 2 Corresponding author, e-mail: ganeev.il11@rambler.ru Acta agriculturae Slovenica, 115/2, 261-271, Ljubljana 2020 115-2 vsebina.indd 261 23. 06. 2020 07:25:17 I. GANEEV et al. 1 introduction In agricultural practice, small seed oil crops such as rapeseed, mustard, and saffron milk, which are in great demand for agriculture and industry, are becoming increasingly important (Hakansson et al., 2013; Kovaly-shyn, 2015; Kovalyshyn et al., 2015). One of the most important operations of oilseed cultivation technology is post-harvest seed treatment (Hakansson et al., 2013; Soares et al., 2016; Moreno et al., 2017). After ripening, rapeseed has a moisture content that amount from 14 to 27 %, and the recommended humidity for storage should be no more than 7 to 8 %. Due to the high humidity in the seeds of oilseeds, oxidative processes can begin, leading to a decrease in their quality. Therefore, timely drying will allow to maintain high sowing and technological qualities of seeds (Ganeev et al., 2009; Ganeev & Masali-mov, 2009; Masalimov et al., 2018). Due to the lack of special technological equipment, drying of oilseed grain is performed on grain drying equipment, which are distinguished by type and method of action (Gabitov et al., 2018). Existing drying methods are mainly based on thermal effects on the material, which in turn leads to a deterioration in the quality of the finished product (Jokiniemi & Ahokas, 2014; Skakov, Rakhadilov & Sheffler, 2013). The choice of drying equipment must be made taking into account the physical and mechanical parameters of a particular culture. It is necessary to classify them to determine the most optimal construction of dryers. Drying devices can be classified according to a number of features, the main ones being the method of supplying heat, the construction of the drying chamber, the operating mode, the state of the grain layer and the construction (GOST, 2008; Sutjagin et al., 2017). The most widespread is the direction of drying grain using the convective method of heat supply. The convective grain dryers are simple and most productive (Soares et al., 2016; Maier, 2017; Manikantan et al., 2018). However, these grain dryers are characterized by high metal consumption, high cost and high energy costs (Sutjagin et al., 2017). In shaft type grain dryers operating on gaseous and liquid fuels, when drying food and industrial grain crops, the regulated specific energy consumption should not exceed 4.56 MJ kg-1 of evaporated moisture, and 5.74 MJ kg-1 when drying seed grain (GOST, 2008). In practice, convective drying devices that operate without heat recovery consume up to 6 MJ kg-1 of evaporated moisture due to the fact that most of the thermal energy is carried away irrevocably by the drying agent. On the territory of the Russian Federation, the drum and shaft grain dryers with convective heat supply are most common, which, when drying the seed, often lead to grain injury. In addition, in shaft dryers, grain is often subjected to local overheating, which in turn leads to protein denaturation in the germ (Shizhuang et al., 2017). The main significant drawback of convective grain dryers is the high energy costs due to the occurrence of a temperature gradient in the material being dried, which leads to a decrease in the drying rate (Jokiniemi et al., 2015). One of the solutions to this drawback is the differentiation of the supply of thermal energy consisting in the alternation of heating the material with its cooling (Jokiniemi et al., 2015). With the development of technology and technics, recently drying methods such as microwave and thermos-radiation methods have begun to spread, the feature of which is the penetration of electromagnetic waves into the depth of the material being dried. This leads to heating of the inner part of the grain bypassing the outer layer (Rogov, 1988). During infrared drying (thermal radiation), the rays are absorbed by the product, which ensures a more uniform heating of the material in depth compared to convective drying (Rogov, 1988). This, in turn, leads to a decrease in the temperature gradient and direct transfer of steam from inside to outside under the influence of the gradient of total pressure (Darvishi et al., 2013; Bet-tega et al., 2014; Zhao et al., 2017). It should be borne in mind that increasing the temperature of infrared heating can lead to damage to the grain germ. In turn, a forced decrease in the heating temperature leads to a decrease in the drying rate and, as a consequence, to an increase in the duration of the drying process, a decrease in the productivity of the dryer, and an increase in energy consumption (Rogov, 2015; Karimov et al., 2016; Martynov et al., 2018). The microwave drying is characterized by internal heating of the material (Li et al., 2014; Zhao et al., 2017). Therefore, thermal diffusion of moisture, directed from the center to the surface of the body, increases the speed of microwave drying. However, in the case of microwave drying of grain, in the absence of temperature and moisture control inside the grain, the probability of germ death due to possible local overheating is high (Ganeev, 2011; Fajzrahmanov et al., 2014; Fajzrahmanov, 2015). At the moment, the problem of the dependence of grain temperature in the inner layers on the power of electromagnetic radiation from microwave is poorly studied. Therefore, the most promising direction is the development of a drying device based on microwave heating of the material and conducting experimental research to identify the operating parameters of the installation, which allows to obtain a high-quality finished product. The purpose of the study is to increase the 262 Acta agriculturae Slovenica, 115/2 - 2020 115-2 vsebina.indd 262 23. 06. 2020 07:25:18 Intensification of the drying process of small seed oilseeds using microwave electromagnetic radiation drying efficiency of small seed oilseeds by applying microwave electromagnetic radiation. 2 methods The choice of the type of dryer and drying method for a particular material is impossible without taking into account its physical and thermophysical properties. In addition, the correct use of the laws of heat and moisture transfer is necessary to determine the most suitable drying mode. The drying process is characterized by internal and external moisture transfer. The optimal combination of technological methods used to increase internal and external moisture transfer will significantly intensify the drying process. The kinetics of moisture transfer in capillary-porous colloidal bodies, which include seeds of agricultural crops, is generally determined by the difference in its potentials (temperature, moisture content). The intensity of internal moisture transfer is described by the well-known equation of non-isothermal moisture conductivity: (1) where is the density of the moisture conduction flux, kg (m-2 ■ h-1); is the flux density of thermal moisture conductivity, kg (m-2 ■ h-1); is the moisture diffusion coefficient in the grain, m2 s-1; is the density of absolutely dry grain, kg m-3; is the thermogradient coefficient, 1 K-1; and are the gradients of concentration (moisture content) and temperature, kg . (kg-' m-1) and K m-1. 1 °moist v ° dry matter ' In this equation, the first term characterizes the movement of moisture in the material under the influ- ence of a moisture gradient, and the second - under the influence of a temperature gradient. When convectively dried, the heat from the upper layers of the material is transferred to the inside, therefore, the gradientsandhave opposite signs, i.e. thermal moisture conduction impedes the advancement of moisture from the surface of the material to its surface (Fajzrahmanov, 2015). As can be seen from equation (1), by reducing the inhibitory effect of thermal moisture conduction or by increasing the flow of moisture conduction, the intensity of internal moisture transfer can be increased. The moisture flow can be increased by increasing the moisture gradient. This can be achieved by exposing the material to electromagnetic radiation in the microwave range (Ganeev, 2011). During microwave heating, the humidity and temperature gradients have the same orientation, and in this case, moisture is removed not only under the influence of thermodynamic forces, but also under the influence of excess pressure arising inside the material (Budnikov, 2008): 1m = -amP3.0.C0;2-4 Frusciante, L., Carli, P., Ercolano, M.R., Pernice, R., Di Matteo, A. (2007). Antioxidant nutritional quality of tomato. Molecular Nutrition & Food Research, 51(5), 609-617. https://doi. org/10.1002/mnfr.200600158 Haberal, M., Aksoy Korpe, D., Darcansoy Içeri, O., Sahin, F.I. (2016). Grafting tomato onto tobacco rootstocks is a practical and feasible application for higher growth and leafing in different tobaccoa tomato unions. Biological Agriculture & Horticulture, 32(4), 248-257. https://doi.org/10.1080/01448 765.2016.1169218 Heinonen, I.M. (2002). Antioxidants in Fruits, Berries and Vegetables: An Overview, in Fruit and Vegetable Processing. 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Effect of Storage Temperatures on Antioxidant Capacity and Aroma Compounds in Strawberry Fruit. Journal of Food Science and Technology, 37(7), 687-695. https://doi. org/10.1016/j.lwt.2004.03.002 Acta agriculturae Slovenica, 115/2 - 2020 305 115-2 vsebina.indd 305 23. 06. 2020 07:25:23 115-2 vsebina.indd 306 23. 06. 2020 07:25:22 doi:10.14720/aas.2020.115.2.1317 Original research article / izvirni znanstveni članek Estragole-rich essential oil of summer savory (Satureja hortensis L.) as an eco-friendly alternative to the synthetic insecticides in management of two stored-products insect pests Asgar EBADOLLAHI 1 2 Received October 29, 2019; accepted March 22, 2020. Delo je prispelo 29. oktobra 2019, sprejeto 22. marca 2020 Estragole-rich essential oil of summer savory (Satureja hortensis L.) as an eco-friendly alternative to the synthetic insecticides in management of two stored-products insect pests Abstract: The lesser grain borer [Rhyzopertha dominica (Fabricius, 1792)] and the red flour beetle [Tribolium castaneum (Herbst, 1797)] are among the cosmopolitan damaging pests on several stored-products. The overuse of chemical pesticides in the control of such pests caused several side-effects including environmental contaminations, human health problems, and insect pests' resistance. In this circumstance, researchers have focused on safe and effective alternatives to chemical pesticides. In the present study, the insecticidal efficiency of essential oil extracted from the summer savory (Satureja hortensis L.) was assessed on the R. dominica and T. castaneum adults. The chemical profile of essential oil was evaluated through a gas chromatography-mass spectrometer, in which estragole, p-ocimene and d-limonene were the main components. The essential oil had considerable fumigant toxicity on insect pests. The mortality of insects was dependent on the essential oil concentration and exposure time. Probit analysis indicated that R. dominica with low LC50 values (Lethal Concentration to kill 50 % of tested insects) was more susceptible than T. castaneum. Accordingly, S. hortensis essential oil with a high level of phe-nylpropanoid and terpenic compounds can be recommended as an efficient and natural alternative to the detrimental chemicals in the management of R. dominica and T. castaneum. Key words: essential oil; estragole; Satureja hortensis; fumigation; coleopteran pests Na estragolu bogato eterično olje vrtnega šetraja (Satureja hortensis L.) kot okolju prijazna alternativa sintetičnim insek-ticidom pri zatiranju dveh vrst skladiščnih škodljivih žuželk Izvleček: Žitni kutar [Rhyzopertha dominica (Fabricius, 1792)] in rižev mokar [Tribolium castaneum (Herbst, 1797) sta kozmopolitski vrsti škodljivcev, ki povzročata škodo na mnogih uskladiščenih pridelkih. Prekomerna raba insekticidov pri zatiranju takšnih škodljivcev ima številne stranske učinke, vključno z onesnaževanjem okolja, zdravstvenimi problemi ljudi in odpornostjo škodljivih žuželk. V tej raziskavi so se raziskovalci osredotočili na varno in učinkovito alternativo sintetičnim in-sekticidom. Insekticidna učinkovitost eteričnega olja iz vrtnega šetraja (Satureja hortensis L.) je bila preizkušena na odraslih osebkih obeh vrst zgoraj omenjenih škodljivcev. Kemična sestava eteričnega olja je bila ovrednotena s plinskim kromato-grafom in masnim spektrometrom, ugotovljeno pa je bilo, da so estragol, p-ocimen in d-limonen glavne sestavine. Zaplinje-vanje z eteričnim oljem je imelo znaten toksični učinek na škodljivi žuželki. Smrtnost žuželk je bila odvisna od koncentracije eteričnega olja in časa izpostavitve. Analiza Probit je pokazala, da je vrsta R. dominica z manjšimi LC50 vrednostmi bolj občutljiva kot vrsta T. castaneum. Glede na to bi lahko eterično olje iz vrtnega šetraja z veliko vsebnostjo fenilpropanoidov in terpenov priporočili kot učinkovito in naravno alternativo škodljivim kemikalijam pri zatiranju omenjenih škodljivcev. Ključne besede: eterično olje; estragol; Satureja hortensis; zaplinjevanje; škodljivi hrošči 1 University of Mohaghegh Ardabili, Moghan College of Agriculture and Natural Resources, Ardabil, Iran 2 Corresponding author, e-mail: ebadollahi@uma.ac.ir Acta agriculturae Slovenica, 115/2, 307-314, Ljubljana 2020 115-2 vsebina.indd 307 23. 06. 2020 07:25:24 A. EBADOLLAHI 1 introduction 2 materials and methods Secondary metabolites announce the evolution of chemical defenses in plants which are often formed as by-products throughout the production of primary metabolites. Secondary metabolites have several essential roles especially in the protection against herbivores and in the attraction of pollinators' (Dinan, 1995; Bohinc et al., 2012). Plant-derived essential oils as well-known secondary metabolites can be produced in several aerial parts including leaves, flowers, seeds, stems and the roots of aromatic plants. Essential oils are generally composed of isoprene units as terpenes and phenylpropane (Bak-kali et al., 2008). Although terpenes such as monoter-penes (2 units of isoprene, C ), sesquiterpenes (3 units of isoprene, C15), and diterpenes (4 units of isoprene, C20) have a high quantity, the monoterpenoids (oxygenated monoterpenes) are often the most components of the many essential oils (Breitmaier, 2006; Abdel-Tawab, 2016). Along with the application of essential oils in the perfumery and pharmaceutical industries, their lethal and sub-lethal effects especially fumigant toxicity of essential oils have been approved toward different class and orders of main insect and acari herbivores (Regnault-Roger et al., 2012; Rojht et al., 2012; Ebadollahi & Jalali-Sendi, 2015). Summer savory [Satureja hortensis L. (Lamiaceae)], as an aromatic spice and food preservative, widely distributed and/or cultivated in many countries. It used in Iranian traditional medicine to treat intestinal and stomach disorders such as indigestion and diarrhea, muscle pain, thrombosis, and cardiovascular diseases (Hajhashemi et al., 2000; Yazdanparast et al., 2008). Moreover, along with antibacterial, antifungal, antioxidant, and cytotoxic activities of S. hortensis, its potential on the insect pest management have also been documented (Mahboubi & Kazempour, 2011; Miladi et al., 2013; Gombac & Trdan, 2014; Farzaneh et al., 2015; Ghorbanpour et al., 2016). R. dominica (lesser grain borer) and T. castaneum (red flour beetle) are among the cosmopolitan serious pests of stored-products such as cereal and legume grains, dried fruits, spices, flours, leather, and even packaging materials made from wood and paper. Further, the quality of infested products strongly reduces due to the residues of insect bodies and their unpleasant smell (Villaverde et al., 2007; Edde, 2012). As part of a program aimed at studying the insecti-cidal activity and chemical composition of plant essential oils, we have assessed the fumigant toxicity and chemical profile of S. hortensis essential oil against R. dominica and T. castaneum. Hope the range of introduced active bioagents derived from aromatic plants has extended by the results of the present study. 2.1 ESSENTIAL OIL EXTRACTION AND ANALYSIS Fresh 10 cm aerial parts from the shoots of S. hortensis were sampled for essential oil extraction. The specimens were collected during April and May 2019 from Parsabad region (Latitude: 39°38' N, Longitude: 47°52' E, and height: 52 m), Ardebil province, Iran. The samples were dried at room temperature within a week and then ground using an electric grinder. Fifty grams ground plant material was poured into a Clevenger apparatus equipped with a 1000 ml balloon. The essential oil was extracted within 3 h and the obtained oil was stored in a refrigerator at 4 °C. Chemical profile of the S. hortensis essential oil was assessed using a gas chromatographic system (Agilent model 7890B) equipped with the mass spectrometer detector (Agilent model 5977A) according to Ebadollahi et al. (2017): chromatographic separation was performed on the HP-5MS (5 % phenyl-methyl-polysiloxane) capillary column (30 m length, 0.25 mm internal diameter, and 0.25 ^m film thickness) with 70 eV ionization energy. The injected volume was 1.0 ^l with 280 °C temperature. The temperature program of the column was set from 50 to 350 °C. Helium (99.999 %) was used as a carrier gas at 1 ml minute-1. The component was identified by comparison of their mass spectra with those from Wiley's MS library (7th edition) and NIST (National Institute of Standards Technology) in the library. 2.2 TESTED INSECTS The adult insects of R. dominica were obtained from the colonies at the Department of plant protection, University of Mohaghegh Ardebili, Ardabil, Iran. The adult insects of T. castaneum were collected from contaminated wheat grains in the warehouses of Parsabad city (Latitude: 39°38' N, Longitude: 47°52' E, and height: 52 m), Ardabil province, Iran. Adult insects were separately released on wheat grains in the breeding container. Adult insects were removed 48 h later and grains with insects' eggs were kept in an incubator at 25 ± 2°C and 65 ± 5 % relative humidity in dark (Arnaud et al., 2005). Synchronized adult insects with 1 - 7 old-days were selected. 2.3 BIOASSAY The fumigation bioassay was done according to the study of Ebadollahi (2018): twenty adults of both insects 308 Acta agriculturae Slovenica, 115/2 - 2020 115-2 vsebina.indd 308 23. 06. 2020 07:25:24 Estragole-rich essential oil of summer savory (Satureja hortensis L.) as an eco-friendly ... of two stored-products insect pests were separately located in 340 ml fumigant chambers. The tested concentrations of essential oil, based on the preliminary experiments, were from 11.76 to 47.06 (l l-1 and from 21.00 to 55.15 (l l-1 for R. dominica and T. cas-taneum, respectively. The essential oil concentrations were poured on the 2 x 3 cm piece of filter papers which were sealed to the inside of the container lids and the lids were closed using parafilm. Experiments were conducted for control groups without adding essential oil concentration. Each treatment was repeated 4 times and the insects' mortality was documented after 24, 48 and 72 h intervals. 2.4 STATISTICAL ANALYSIS Variance analysis was used to assess the significant effects of essential oils' concentrations and the exposure times. To compare the effects of independent factors concentration and exposure time on the insects' mortality, the w2 comparison was used. Calculation of lethal concentrations (LC), lethal times (LT) and linear regression analysis along with heterogeneity of the data by a Chi-squared test were done using SPSS software version 24 (IBM, Chicago, USA). 3 results 3.1 CHEMICAL COMPOSITION OF ESSENTIAL oIL Chemical analysis of S. hortensis essential oil identified 17 components at 99.21 %, in which 83.02 % are phenylpropanoid constituents. Five different groups of terpenes were also recognized in the essential oil, in which the monoterpene hydrocarbons (15.38 %) had the highest amount followed by sesquiterpenoids (0.43 %), monoterpenoids (0.26 %), a sesquiterpene hydrocarbon (0.08 %), and a diterpene (0.04 %). Estragole (82.10 %) as Table 1: Chemical composition of the essential oil isolated from Iranian Satureja hortensis Compound Retention Time (minute) Formula and Classification Percentage a-Pinene 5.30 C10H MH 0.91 Camphene 5.57 C10H MH 0.04 Sabinene 6.03 C10H MH 0.06 ß-Pinene 6.09 C10H MH 0.09 ß-Myrcene 6.33 C10H MH 0.12 d-Limonene 7.08 C10H MH 2.25 ß-Ocimene 7.46 C10H MH 11.86 a-Terpinene 8.27 C10H MH 0.05 Rosefuran 8.43 C10H 4o M 0.08 Estragole 11.51 C10H 2o Ph 82.10 E,E-2,6-Dimethyl-3,5,7-octatriene-2-ol 11.54 C10H 6o M 0.07 Bornyl acetate 14.23 C12H 60o2 M 0.11 Methyl Eugenol 18.71 C11H 4o2 Ph 0.92 Germacrene-D 21.05 C15H SH 0.08 spathulenol 23.76 C15H 40 S 0.31 Caryophyllene oxide 23.89 C15H 40 S 0.12 Eicosane 32.78 C20H DH 2 0.04 MH: Monoterpene Hydrocarbon 15.38 M: Monoterpenoid 0.26 SH: Sesquiterpene Hydrocarbon 0.08 S: Sesquiterpenoid 0.43 DH: Diterpene Hydrocarbon 0.04 Ph: Phenylpropanoid 83.02 Total 99.21 Acta agriculturae Slovenica, 115/2 - 2020 309 115-2 vsebina.indd 309 23. 06. 2020 07:25:24 A. EBADOLLAHI Table 2: Results of the variance analysis of S. hortensis essential oil fumigation on the adults of R. dominica and T. castaneum after 24, 48 and 72-h exposure times Insect Source of Variation df F p-value w2 R. dominica Concentration 4 467.987 * <0001 22.516 Time 2 155.009 * <0001 3.713 Time x Concentration 8 1.594 0.154 0.057 T. castaneum Concentration 4 324.572 * <0001 17.793 Time 2 142.271 * <0001 3.884 Time x Concentration 8 1.106 0.377 0.012 Significant at a = 1% Figure 1: Concentration - mortality lines for fumigant toxicity of S. hortensis essential oil against the adults of R. dominica and T. castaneum after 24, 48 and 72-h exposure times a phenylpropanoid constituent had the highest amount and monoterpene hydrocarbons ^-ocimene (11.86 %), and dl-limonene (2.25 %) were in the next points (Table 1). 3.2 FUMIGANT TOXICITY Results of the fumigant toxicity indicated that essential oil of Iranian S. hortensis had considerable toxicity on the R. dominica and T. castaneum adults. The results of variance analysis were summarized in Table 2. Concentrations of essential oil and exposure times had statistically significant effects on the insects' mortality but 310 Acta agriculturae Slovenica, 115/2 - 2020 their interaction wasn't significant. Furthermore, based on the w2 values, among these factors, the effect of essential oil concentration was more effective. The calculated R2 values for concentrations-mortality correlation were 0.959, 0.935 and 0.940 for R. dominica and 0.956, 0.953 and 0.960 for T. castaneum after 24, 48 and 72-h exposure times, respectively. So, there is a direct correlation between the concentrations of essential oil and mortality of both insects (Figure 1). Probit analysis indicated the calculated LC50 values (lethal concentration to kill 50 % of tested insects) of essential oil were significantly decreased from 24 h to 72 h for both insects (Table 3). For example, the 24 h-LC50 value of essential oil with 95 % confidence limits was 27.212 115-2 vsebina.indd 310 23. 06. 2020 07:25:24 Estragole-rich essential oil of summer savory (Satureja hortensis L.) as an eco-friendly ... of two stored-products insect pests Table 3: Results of Probit analysis for fumigant toxicity of S. hortensis against the adults of R. dominica and T. castaneum Time LC50 (95 % confidence limits) x2 Insect (h) (pl l-1) (df = 3) Slope ± SE Significance * R. dominica 24 27.212 (24.657 - 30.361) 3.893 2.740 ± 0.294 0.273 48 22.193 (20.140 - 24.385) 7.062 2.897 ± 0.298 0.070 72 16.466 (12.128 - 20.013) 5.830 3.321 ± 0.329 0.120 T. castaneum 24 38.908 (35.951 - 42.688) 3.425 3.386 ± 0.412 0.331 48 30.757 (28.377 - 33.070) 3.810 3.691 ± 0.419 0.283 72 25.747 (23.020 - 28.021) 2.745 3.506 ± 0.429 0.433 Concentration LT50 (95 % confidence limits) X2 Insect (pl l-1) (h) (df = 1) Slope ± SE Significance * R. dominica 47.06 10.301 (2.944 - 16.210) 1.765 2.060 ± 0.515 0.184 T. castaneum 55.15 12.682 (5.479 - 18.103) 2.023 2.282 ± 0.503 0.155 Since the significance level is greater than 0.05, no heterogeneity factor is used in the calculation of confidence limits. The number of insects for (24.657 - 30.361) pl l-1 which was decreased to 16.466 (12.128 - 20.013) pl l-1 after 72 h. Further, according to Table 3, adults of R. dominica with low LC50 values were significantly susceptible than T. castaneum adults to the S. hortensis essential oil at all exposure times. The lethal times to kill 50 % of tested insects (LT50 values) are also shown in Table 3. At a high tested concentration of S. hortensis essential oil (47.06 pl l-1), the LT50 value was 10.301 (2.944 - 16.210) h against R. dominica adults. This value for T. castaneum adults with a concentration of 55.15 pl l-1 was calculated as 12.682 (5.479 - 18.103) h. 4 discussion The composition of S. hortensis essential oil have been investigated in the previous studies; carvac-rol (11.0 %), p-cymene (19.6 %), sabinene (4.4 %), y-terpinene (16.0 %), and thymol (28.2 %) were found as major compounds by Mahboubi and Kazempour (2011). Thymol, p-cymene, y-terpinene, and carvacrol were not detected in the present study but a trace of sabi-nene (0.06 %) was determined. In contrast, estragole and ^-ocimene as major components of present work were not detected in the study of Mahboubi and Kazempour (2011). Farzaneh et al. (2015) showed carvacrol (48.0 %), p-cymene (11.7 %), myrcene (2.5 %), a-pinene (2.5 %), y-terpinene (24.2 %) were the main components. From these constituents, myrcene (0.12 %) and a-pinene (0.91 %) with different amounts were recognized in the essential oil of present study. In the other study, Miladi et al. (2013) also revealed that monoterpenoids (59.11 %) were the main chemical class of S. hortensis essential oil which is parallel with our results but they announced other components such as carvacrol, ^-caryophyllene, p-cymene, and y-terpinene. In contrary, Mohammadhos-seini and Beiranvand (2013) showed that the monoter-pene hydrocarbons such as myrcene, a-pinene, ^-pinene, a-terpinene, and a-thujene had the highest amount in the S. hortensis essential oil. These differences in the chemical profile of S. hortensis essential oil in the present and above-mentioned studies can be due to the variations in some of the influential factors, such as geographical and growing conditions, drying and extraction methods, on-togenetic stages, and season (Sefidkon et al., 2006; Pfefferkorn et al., 2008; Rezvanpanah et al., 2011; Ghorban-pour et al., 2016). Insecticidal properties of S. hortensis essential oil were acknowledged in some recent studies; appropriate fumigant toxicity of this oil was proved against Mediterranean flour moth [Ephestia kuehniella (Zeller, 1879)], Indianmeal moth [Plodia interpunctella (Hubner, 1813)], and T. castaneum (Mollaei et al., 2011). The calculated 48 h-LC50 value for T. castaneum in this work (192.350 pl l-1) is much higher than the corresponding LC50 in the present study (30.757 pl l-1). In the study of Tozlu et al. (2011), the S. hortensis essential oil with a high amount of carvacrol, ^-caryophyllene, p-cymene, S-terpinene, and a-terpinene was very toxic against the broad bean weevil [Bruchus dentipes (Baudi, 1886)]. They concluded that the S. hortensis essential oil toxicity is directly related to its components. Along with the fumigant toxicity of S. hortensis essential oil, the contact toxicity, repellency, and disruption in the enzymes' activity were also described (Mollaei et al., 2011; Heydarzade & Moravvej, 2012; Magierowicz et al., 2019). The results of these studies indicated that S. hortensis essential oil has considerable Acta agriculturae Slovenica, 115/2 - 2020 311 calculation of LC values is 400 for each time. The number of insects for calculation of LT values is 240 for each concentration. 115-2 vsebina.indd 311 23. 06. 2020 07:25:24 A. EBADOLLAHI insecticidal activities against stored-product insect pests which are in accordance with our findings. Estragole or methyl chavicol, as two major compounds identified in the present study, is a GRAS (Generally Recognized As Safe) nominated material and approved for food procedure (De Vincenzi et al., 2000). Its name originates from "estragon" which is a French word of tarragon (Artemisia dracunculus L.) (Misztal et al., 2010). Along with cytotoxic and antimicrobial properties of estragole (Bagamboula et al., 2004; Andrade et al., 2015), toxicity of this compound has also been approved against some of damaging stored-product insect pests including T. castaneum, the rice weevil [Sitophilus oryza (Linnaeus, 1763)], the maize weevil [Sitophilus zeamais (Motschulsky, 1855)], the booklice [Liposcelis bostry-chophila, Badonnel, 1931], the cigarette beetle [Lasio-derma serricorne (Fabricius, 1792)], and the adzuki bean beetle [Callosobruchus chinensis (Linnaeus, 1758)] (Kim & Ahn, 2011; Wang et al., 2011; Kim & Lee, 2014; Guo et al., 2015). Furthermore, the insecticidal properties of other main components identified in the present study including d-limonene and ^-ocimene were also documented (Tripathi et al., 2003; Guo et al., 2015; Kang et al., 2018). Accordingly, the fumigant toxicity of S. hortensis essential oil may be attributed to such constituents. However, the existence of synergistic effects between other compounds is also possible. 5 conclusion Synthetic pesticide residues can be found in different parts of our surrounding environment from water and soil to everybody's foods and even human breast milk samples (Damgaard et al., 2006; Nicolopoulou-Stamati et al., 2016; Trdan, 2016). Regarding the pests' management, due to the overusing of synthetic chemicals, the other side-effects such as resurgence and outbreak of new pests, several pest-resistant reports on the different classes of synthetic pesticides, and detrimental effects on valuable noun-target organisms including parasitoids and predators have also been documented (Kohler et al., 2013; Cruz et al., 2017; Sudo et al., 2018). Therefore, urgent efficacious tools for the reduction of synthetic chemical utilization and for announcing eco-friendly agents with fewer public health risks are required. Because of the low toxicity to the mammals and pose a minimum risk, the plant essential oils considered safe (Viciolle et al., 2012). The prospective pesticidal activity of several plants essential oils have been stated in recent years (Isman & Grieneisen, 2014), and the range of these eco-friendly bio-agents was extended in the present study through the introduction of Iranian phenyl- propanoid-rich summer savory as a toxic agent against two damaging coleopteran insect pests R. dominica and T. castaneum. However, based on the short residual lifetime (Isman, 2006), it is recommended that such essential oils be tested in the better applicable form such as "controlled release technique" through micro- and nano-encapsulation. 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Vascular Pharmacology, 48, 32-37. https://doi.org/ 10.1016/j.vph.2007.11.003 314 Acta agriculturae Slovenica, 115/2 - 2020 115-2 vsebina.indd 314 23. 06. 2020 07:25:24 doi:10.14720/aas.2020.115.2.1257 Original research article / izvirni znanstveni članek Improvement of yield and yield stability in safflower using multivariate, parametric and non-parametric methods under different irrigation treatments and planting date Pooran GOLKAR1' 2 3, Nasrin RAHMATABADI 4, Seyyed Ali Mohammad MIRMOHAMMADY MAIBODY 4 Received September 01, 2019; accepted March 31, 2020. Delo je prispelo 01. septembra 2020, sprejeto 31. marca 2020 Improvement of yield and yield stability in safflower using multivariate, parametric and non-parametric methods under different irrigation treatments and planting date Abstract: Development of superior genotypes with high adaptability to different environments is considered as one of the most important goals in safflower breeding programs. In this study, ten parametric and six non-parametric measures along with the additive main effects and the relevant multiplicative interaction (AMMI) model were used to evaluate genotype by environment interaction (GE) in 15 safflower genotypes across 12 test environments ) combination of year, planting date and moisture conditions) during growing seasons in 2016 and 2017. AMMI analysis revealed significant differences among the genotypes and their GE interactions. The different stability statistics were substantiated by rank correlation coefficient. Rank-correlation coefficients revealed positive and significant correlations between mean seed yield and superiority index (r = 0.99**), and significant and negative correlation with bi, R2, D.j and non- parametric measures (NPi(2), NPi(3) and NPi(4)). Based on most stability parameters, the Mex.295 genotype (G) was found to be the most stable for seed yield. IL.111 genotype (G9) recorded the highest mean yielding genotype regarded as the most favorable safflower genotype. In conclusion, both stability and seed yield should be simultaneously considered to exploit useful effects of G x E interactions in safflower breeding programs. Key words: safflower; parametric and non-parametric measures; yield, rank correlation Izboljšanje pridelka žafranike in njegove stabilnosti z multi-variatnimi parametričnimi in neparametričnimi metodami pri različnem namakanju in datumih setve Izvleček: Razvoj superiornih genotipov z veliko prilagodljivostjo različnim okoljem je eden izmed najvažnejših ciljev v žlahniteljskih programih žafranike. V raziskavi je bilo uporabljenih deset parametričnih in šest neparametričnih meril vključno z glavnimi aditivnimi učinki in modelom pomembnih multiplikativnih interakcij (AMMI) za ovrednotenje interakcije genotipa z okoljem (GE) pri 15 genotipih žafranike, preiskušenih v 12 okoljih )kombinacija leta poskusa, datuma setve in vlažnostnih razmer) v rastnih sezonah 2016 in 2017. AMMI analiza je odkrila značilne razlike v interakcijah genotipov z okoljem. Različne statistične metode za ovrednotenje različnih vidikov stabilnosti pridelka so bile uspešno nadomeščene s koeficientom gradualne korelacije. Ti koeficienti so odkrili pozitivne in značilne korelacije med poprečnim pridelkom semena in indeksom superiornosti (r = 0.99**), in značilne negativne korelacije z bi, R2, D^ in neparametričnimi merili (NPi(2), NPi(3) in NPi(4)). Na osnovi večine parametrov stabilnosti je bil genotip Mex.295 ,(G ) prepoznan kot najbolj stabilen za pridelek semena. Genotip IL.111 (G9) je bil prepoznan kot najboljši genotip žafranike z največjim poprečnim pridelkom. Zaključimo lahko, da je v žlahtniteljskih programih žafranike potrebno hkrati upoštevati velikost in stabilnost pridelka, če hočemo izkoristiti koristne interakcije okolja in genotipa (G x E). Ključne besede: žafranika; parametrična in ne-parametrična merila; pridelek; korelacija rangov 1 Isfahan University of Technology, Department of Natural Resources, Iran 2 Isfahan University of Technology, Research Institute for Biotechnology and Bioengineering, Iran 3 Corresponding author, e-mail: poorangolkar@gmail.com, golkar@ iut.ac.ir 4 Isfahan University of Technology, Department of Agronomy and Plant Breeding, College of Agriculture, Iran Acta agriculturae Slovenica, 115/2, 315-327, Ljubljana 2020 115-2 vsebina.indd 315 23. 06. 2020 07:25:25 P. GOLKAR et al. 1 introduction Safflower (Carthamus tinctorius L.) is mainly grown in dryland conditions of the world as an oilseed crop with diverse genetic backgrounds and the pharmaceutical industry uses (Kumar et al., 2016). Safflower have tremendous potential for cosmetic industry and organic food and other usages as biofuel, soap, varnish making, food coloring, flavoring, dyes, medicines and bird feed (Golkar, 2014; Kumar et al., 2016). With the development of global changes, researchers from all over the world increasingly pay attention to drought as a major abiotic stress limiting growth and productivity of crops. Iran is known as one of the highest genetic diversity for safflower in the world (Knowles, 1969). It hosts a large number of native landraces with improved yields in seed and oil (Golkar, 2014). Drought can be regarded, as a major fundamental abiotic stress factor limiting and restricting the crop plants growth and production (Farooq et al., 2012 Hus-sain et al., 2016). So, drought stress has recently attracted increasing attention in breeding programs due to its exacerbating impact of it by climate change (Hussain et al., 2016). In drought affected regions and semi- arid agro-ecosystems, safflower is considered as a promising alternate crop due to its high adaptability to drought conditions (Omidi Tabrizi, 2006; Kar et al., 2007; Hus-sain et al., 2016). The yield of safflower is influenced by such different factors as location and date of planting, soil available water, air temperature, and light intensity (Da-jue & Mundel, 1996), especially during its seedling and flowering stages (Hussain et al., 2016). Different environments usually have significant fluctuation on seed yield of different genotypes due to the different responses of the genotypes to environmental features including environmental stresses (biotic and abiotic). Hence, seed yield is influenced by genotype (G), environment (E) and genotype x environment interactions (GxE) in a number of genotypes that are grown in a wide range of environments (Gauch, 2006). The seed yield of safflower genotypes varies a lot due to the high dependence of their yield on both genotypic and environmental conditions (Omidi Tabrizi, 2006; Ebrahimi et al., 2016). In safflower breeding programs, interpretation of G x E interactions plays a major role to identify the superior genotypes across various environments (Pourdad & Mo-hammadi, 2008). Also, the obtained results from G x E analysis determine the phenotypic stability of genotypes in each tested environment (Abdulahi et al., 2009). In such situations, the breeder is often faced with the choice either to develop some special genotypes for a specific adaptations and/or to choose the genotypes with a high general adaptations that can perform well in a wide range of environments (Pourdad & Mohammadi, 2008) Thus, it is necessary to study the adaptability and stability of new genotypes with diverse origins for cultivation in different planting dates and moisture regimes in its cultivation regions as Iran. Different methods have been commonly used to determine the extent of G x E interaction effects under different growing conditions (Becker & Leon, 1988). These methods include multivariate analysis (Gauch, 2006), parametric methods (Eberhart & Russell, 1966), and non-parametric ones (Thennarasu, 1995). Parametric methods, as the most common approach, depend on assumptions made regarding the distributional patterns of about genotypic, environmental, and G x E interaction effects (Huehn, 1996). The most common ones include regression coefficient (b.) (Eberhart & Russell, 1966), regression coefficient (B.) (Perkins & Jinks, 1968), variance of deviations from regression (s2di) (Eberhart & Russell, 1966), Wricke' s ecovalance (W2) (Wricke, 1962), coefficient of variability (CV) (Francis & Kannenberg, 1978), and stability variance () (Shukla, 1972). Most breeding programs exploit combinations of some parametric and some non- parametric approaches (Becker & Leon, 1988). Non-parametric approaches are based on no assumption about the distribution of model residuals and homogeneity of variances (Nassar & Huehn, 1987; Farshadfar et al., 2012). Multivariate techniques have been commonly employed in stability analysis in order to provide more information regarding the real multivariate response of genotypes to different environments (Purchase et al., 2000). Multivariate analysis serves three purposes: (i) to remove noise from the data pattern, (ii) to make a summary of the data, and (iii) to show the structure existing in the data. Additive main effects and multiplicative interactions (AMMI) model combines the main effects and interactions of genotype by environment. This method have its own capacities as identification of the ideal test conditions, choice of genitors, and formulation of recommendations for regionally adapted cultivars (Gauch & Zobel, 1996; Ebdon & Gauch, 2002). The AMMI stability value (ASV) was developed by Purchase et al. (2000) based on the AMMI model scores (IPCA1 and IPCA2) for each genotype. The ability of safflower varieties to function appropriately in different environmental conditions has been well confirmed by plant breeders and agronomists. The present study is intended to identify the potential of native and exotic safflower genotypes for cultivation in arid and semi-arid regions based on the best sowing dates. So, the main objective of this study was to investigate the genotype by environment interactions for the seed yield of safflower genotypes, as evaluated under different environmental conditions (year, sowing date, and moisture regimes) and 2) to find stable safflower 316 Acta agriculturae Slovenica, 115/2 - 2020 115-2 vsebina.indd 316 23. 06. 2020 07:25:25 Improvement of yield and yield stability in safflower using multivariate, parametric and ... under different irrigation treatments and planting date genotypes having high seed yields in a wide range of environments. 2 materials and methods This experiment was conducted in 2016 and 2017 at the Research Farm located at Isfahan University of Technology, in Lavark, Isfahan (32° 32'N, 51° 23' E, 1630 m asl), Iran. The soil at the site is silty clay loam with the pH value of 7.8. In each of the study years, fifteen safflower accessions from various topographical regions (both na- tive and exotic accessions) were planted (Table 1) with three replications at each of the two dates designated as early sowing (15 March) and late sowing (15 April). Plants were irrigated uniformly at the budding stage. The non-stress treatment involved irrigation when 40 % of the total available water was depleted from the root zone. In the medium and high drought stress treatments, irrigation was applied when depletion of 60 % and 85 of the total available water from the root zone occurred, respectively. Irrigation depth was determined using the formulae: I = [(0 - 0 ,)/100] DxB), where, I is the irrigation depth (cm) and 0 FC (-0.03 MPa) denotes the soil Table 1: Safflower genotype origins and the environmental characteristics of the environments used to analyze genotype x environment interaction on safflower seed yield using parametric and nonparametric measures Genotype characteristics Genotype Name Origin Genotype type Mean seed yield (g/plant) G1 AC Sunset Canada - 13.72 G2 KMP30 Karaj, Iran Selected from mutation 15.94 G3 GE62918 Germany - 11.45 G4 Mex.7-37 Mexico - 9.30 G5 KMP51 Karaj, Iran selected from mutation 12.20 G6 C111 Isfahan, Iran selected from landrace 13.43 G7 K21 Kordestan, Iran selected from landrace 9.91 G8 Padideh Isfahan, Iran selected from landrace 12.51 G9 IL.111 Auromieh, Iran selected from landrace 14.46 G10 Mex.295 Mexico Pedigree method 17.22 G11 Mex.117 Mexico - 11.56 G12 A2 Azerbayejan, Iran - 13.24 G13 Gol Sefid Isfahan, Iran selected from landrace 14.77 G14 PI-25090 Turkey - 8.91 G15 Golmehr Isfahan, Iran Zarghan 279 x IL.111 18.42 Environment characteristics Environment Year- Location- Sowing date-Irrigation treatment Mean seed yield /plant (g/plant) E1 2016- Lavark- 15 March- Non-drought stress 18.56 E2 2016- Lavark- 15 March - Medium drought stress (60 % FC) 13.63 E3 2016- Lavark- 15 March- High drought stress (85 % FC) 13.93 E4 2016- Lavark- 15 April- Non-drought stress 12.51 E5 2016- Lavark- 15 April- Medium drought stress (60 % FC) 9.13 E6 2016- Lavark- 15 April- High drought stress (85 % FC) 8.24 E7 2017- Lavark- 15 March- Non-drought stress 21.78 E8 2017- Lavark- 15 March - Medium drought stress (60 % FC) 14.51 E9 2017- Lavark- 15 March High - drought stress (85 % FC) 13.76 E10 2017- Lavark- 15 April- Non-drought stress 13 E11 2017- Lavark- 15 April- Medium drought stress (60 % FC) 9.83 E12 2017- Lavark- 15 Apri- High drought stress (85 % FC) 8.75 Acta agriculturae Slovenica, 115/2 - 2020 317 115-2 vsebina.indd 317 23. 06. 2020 07:25:25 P. GOLKAR et al. gravimetric moisture percentage at field capacity (22 %); on the other hand, 0. (-1.5 MPa) indicates the soil gravimetric moisture percentage at the irrigation time (10 %), D refers to the root-zone depth (50 cm), and B relates to the soil bulk density at the root zone (1.3 g cm-3) (Clarke et al., 2008). The characteristics of the different genotypes and environments used in this study are reported in Table 2. 2.1 STATISTICAL ANALYSIS 2.1.1 Variance analysis For detection the magnitude effects of genotype, environment and genotype x environment, a combined analysis of variance carried out, based in three replication in each environment. The soft ware GEA-R (v.4.1) (Pacheco et al., 2015) were used for all of the calculations. 2.1.2 AMMI analysis The additive main effects as well as multiplicative interaction (AMMI) model were employed according to the following formula (Gauch & Zobel, 1996): where, p represents the grand mean, g refers to the main effect of the ith genotype, and e. denotes the main effect of the jth environment. GEI is captured by: H = 1 In this equation, represents the Eigen value of the nth interaction principal component analysis (IPCA) which is retained in the AMMI model, refers to the eigen vector taken for the ith genotype from the nth IPCA, indicates the Eigenvector considered for the jth environment from the nth IPCA, indicates the GEI residual, n shows the number of IPCA kept in the model and finally, e stands for the random error term. 2.1.3 Parametric statistics high average yields were considered as the most desirable ones. )x100 2) Regression approaches Eberhart and Russell (1966) used the linear regression coefficient (bi) (a part) and pooled deviation mean squares ( (b section) to study the GxE interaction. (a)b = 1 + S; (Xij - Xi. -X.j + X..)( X.j -X..) mi = E/X.J-X..)2 Z[xij-x,.-x.j+x..]-b,2E'iXj-x,j2 si e — 2 r According to Perkins and Jinks (1968), the stable variety in each genotype is defined by small values of D and non-significance of B = 1. Bi = y1J(xi;-xi.-x.j + x..)(x.j-x..) D.. 3) Coefficient of determination (R2) The most stable genotype is characterized by the minimum value of R2 (Pinthus, 1973). R- = (Xij-X..~)z ZjiXij-Xi.)2 Here, X.. represents the safflower yield of the genotype i in the environment j, X. denotes the mean safflower yield of the genotype i, X. stands for the mean safflower yield in the environment j, X.. is the grand mean, b denotes the regression coefficient, e is taken as the number of environments and finally, g indicates the genotypes number. Wricke covalence (W2) W* = sr X- xj x >